Ultrastructure and electrolyte transport of the epithelium of coprodeum, colon and the proctodeal diverticulum of Rhea americana.

The structure and function of the lower intestinal tract of Rhea americana were characterized to evaluate the evolutionary relationship to other struthioniform and avian species. In 5 rheas the gross anatomy and the light and transmission electron microscopy were studied in parallel to in vitro electrophysiological measurements of ion transport. The mucosa in the colon was amplified with villi, often branched, and in the coprodeum with folds. In both tissues the epithelium was a monolayer composed of columnar absorptive cells, goblet cells and mitochondria-rich cells. Colon and coprodeum appeared to produce large amounts of mucus. The proctodeal diverticulum was rich in lymphoid tissue arranged into lobuli bursales, and it was concluded that this structure is a modified bursa of Fabricius. The sparse interlobular epithelium of the diverticulum resembled that of colon and coprodeum. Baseline short circuit currents (I(SC)) averaged 114.5+/-13.8 microA/cm(2) in colon, 193.1+/-30.3 microA/cm(2) in coprodeum and 60.4+/-9.1 microA/cm(2) in the diverticulum. Amiloride sensitive Na+-transport amounted to 31, 88 and 38% of the baseline I(SC) in these three tissues, respectively. In all tissues, there was also a modest, theophylline activated chloride secretion response, and ouabain, the Na+/K+-ATPase inhibitor, abolished most of the I(SC). The transepithelial resistance (TER) of the diverticulum was much higher than the other tissues. Upon dissection, urate from ureteral urine was observed in the lower third of the colon and to a lesser extent in the proctodeal diverticulum, indicating retrograde peristalsis of the urine. Thus, unlike the ostrich, there is no sphincter separating colon and coprodeum. On the other hand, a thick mucus layer was seen overlying the mucosa in both colon and coprodeum, as in the ostrich. This may help to prevent osmotic water loss, despite the presence of hyperosmotic urine (up to 800 mOsm) in the lower intestine. Both morphological and electrophysiological data from the rhea support the hypothesis that the rhea lower intestine contributes to post-renal modification of ureteral urine and to the regulation of osmotic balance, as also seen in domestic fowl and other avian species. The proctodeal diverticulum functions mainly as an immune organ, with only limited transport capability.

beta-Amino acid transport in pig small intestine in vitro by a high-affinity, chloride-dependent carrier.

This study describes unidirectional influx of amino acids and D-glucose across the small intestinal brush-border membrane of fully weaned eight week old pigs. Influx is minimal in the duodenum and maximal in the distal and/or mid small intestine. Influx of beta-alanine, taurine and N-methyl-aminoisobutyric acid is chloride-dependent. The activation stoichiometry for taurine influx is 1.0 +/- 0.2 chloride/2.4 +/- 0.3 sodium/1 taurine. Influx of D-glucose, lysine, glycine and glutamate is chloride-independent. An ABC test demonstrates a common beta-amino acid carrier: (a) the apparent affinity constant K1/2Taurine is 44 +/- 13 microM (means +/- S.D.) and the inhibitory constant (KiTaurine) against beta-alanine influx is 41 +/- 5 microM (means +/- S.E.). (b) K1/2beta-alanine is 97 +/- 23 microM and Kibeta-alanine against taurine influx is 160 +/- 22 microM. (c) KiHypotaurine against taurine and beta-alanine influx is 43 +/- 4 (n = 7) and 22 +/- 5 microM (n = 7), respectively. In conclusion, a high affinity, low capacity, sodium- and chloride-dependent carrier of beta-amino acids is present in pig small intestine.

Chronic aldosterone therapy and the control of transepithelial transport of ions and water by the colon and coprodeum of the domestic fowl (Gallus domesticus) in vivo.

White Leghorn laying hens were maintained on commercial poultry food (medium-Na+ diet) and fresh water. Birds maintained on a high-Na+ diet received, in addition, 10 ml 9% (w/v) NaCl/kg by stomach load for 2 days before the perfusion experiments. Some birds on each dietary Na+ level also received aldosterone injections (60 micrograms/kg per day, i.m.) for 2 days before the perfusion experiments. The lumen of the coprodeum and colon of anaesthetized birds was perfused with solutions resembling ureteral urine, with systematically varied Na+, NH4+, Cl- and osmotic concentrations. Aldosterone enhanced net Na+ absorption (JNa) and associated net Cl- absorption and K+ secretion, and induced (in birds on medium- but not on high-Na+ diets) the appearance of a saturable JNa component dependent on the luminal concentration of Na+. Aldosterone enhanced net absorption of NH4+ and decreased the transmural potential difference in birds on a high-Na+ diet only; water and phosphate fluxes were not affected. Disparities between aldosterone- and Na+-depletion-induced effects suggested that one or more factors (in addition to aldosterone) are involved in the normal mediation of the responses of the lower intestine of the domestic fowl to varied Na+ intake.

Acute effects of aldosterone on water and electrolyte transport in the colon and coprodeum of the domestic fowl (Gallus domesticus) in vivo.

White Leghorn laying hens were maintained on commercial poultry food (medium-Na+ diet) and fresh water. High-Na+ diet birds received, in addition, 10 ml 9% (w/v) NaCl/kg by stomach load for 2 days before perfusion experiments. The lumen of the coprodeum and colon of anaesthetized birds was perfused with a hyperosmotic solution resembling ureteral urine. Transmural solute and water fluxes and potential difference (p.d.) values were measured for 2.5 h before and for 8 h after i.v. injection of aldosterone (120 microgram/kg). After administration of aldosterone, the lag, increasing and stabilized plateau phases were identified for Na+, K+ and Cl- fluxes (which together formed an electroneutral ion-exchange system); plateau flux values were significantly greater than preinjection values and were comparable to values for birds maintained on low-Na+ diets in parallel experiments. Ammonium, phosphate and water fluxes were unresponsive to aldosterone and p.d. values showed a transient increase in medium-Na+ diet birds only. In parallel experiments on birds on low sodium diets the ammonium flux and p.d. increased but the osmotic flow and phosphate transfer did not respond. Therefore acute injection of aldosterone reproduced some but not all of the responses to dietary Na+ restriction in fowls.

Tachykinins mediate changes in ion transport in porcine jejunum through release of prostaglandins and neurotransmitters.

In the present study, we investigated the mediators involved in substance P (SP) and neurokinin A (NKA) induced ion transport. Stripped preparations of porcine jejunal tissue were mounted in Ussing-chambers and short-circuited. The cyclo-oxygenase inhibitor, piroxicam (10 microM) and the neuronal conduction blocker, tetrodotoxin (TTX) (0.1 microM) both significantly decreased the SP (0.1 microM) (66% and 36%, respectively) and NKA (1 microM) (64% and 31%, respectively) induced increase in short-circuit current (SCC). Pretreatment with both piroxicam and TTX totally abolished the SP and NKA response. SP (0.1 microM) caused a significant release of prostaglandin E2 (PGE2), whereas the release of PGE2 induced by NKA was not significant. Experiments were performed to clarify if vasoactive intestinal polypeptide (VIP) was mediating SP or NKA responses. VIP caused a TTX-insensitive and a concentration-dependent increase in SCC. Two VIP antagonists did not change the response to VIP (10 nM and 0.1 microM). Thus, these antagonists could not be used to further elucidate the role of VIP. We were unable to measure a significant release of VIP after SP or NKA treatment. These results indicate, that SP and NKA regulate ion transport in porcine jejunum, entirely through the release of prostaglandins and enteric neurotransmitters.

Secretory pathways in Salmonella Typhimurium-induced fluid accumulation in the porcine small intestine.

The involvement of 5-hydroxytryptamine (5-HT) and 5-HT3 receptors and prostaglandin E2 (PGE2) in Salmonella Typhimurium-induced fluid accumulation in the porcine small intestine was investigated. Salmonella Typhimurium (10(8) and 10(10) cfu) and cholera toxin (CT; 20 microg) were instilled for 8 and 11 h in ligated loops in the porcine jejunum and ileum. Fluid accumulation and concentrations of Na+, K+, Cl-, 5-HT and PGE2 in the fluid accumulated in the loops were measured. The fluid accumulation was also measured when Salmonella Typhimurium (10(10) cfu) and CT (20 microg) were instilled for 8 h in ligated loops in jejunum and ileum in pigs given subcutaneous injections of saline or the 5-HT3 receptor antagonist ondansetron (200 microg/kg). Salmonella Typhimurium (10(10) cfu) and CT both induced fluid accumulation in jejunum and ileum after 8 and 11 h. Both treatments also induced an increase in luminal release of 5-HT and PGE2. The accumulated fluid was iso-osmotic and hyperosmotic in CT- and Salmonella Typhimurium-treated loops, respectively. Ondansetron reduced the Typhimurium-induced fluid accumulation in both jejunum and ileum by c. 40%, while it failed to reduce the response to CT. These results demonstrate that 5-HT and PGE2 are released and 5-HT3 receptors activated in the secretory pathway of Typhimurium in the porcine small intestine.

Advanced computer control of electrophysiological experimentation.

A special configuration of the data acquisition software package Spike2 (CED) has been developed to allow interactive computer control of a current-clamp intracellular recording system. Using the 1401plus dedicated computer (CED) as an interface between the electrophysiological apparatus and a personal computer it was possible to have keyboard-control of intracellular current injection, single and repetitive pulse nerve stimulation, pressure ejection as well as on-going data acquisition. An analysis program was designed using the Spike2 programming language for the investigation of resting membrane properties, spike characteristics and synaptic input profiles of enteric neurones. The hardware configurations and associated software of our set-up may be of interest to electrophysiologists wishing to implement or extend a computer-based experimental system.

Functional characterisation of tachykinin receptors mediating ion transport in porcine jejunum.

In the present study, tachykinin receptors (designated NK 1, NK2 and NK3) involved in regulation of ion transport in porcine jejunum were characterised. Stripped tissue preparations were mounted in Ussing chambers and short-circuited. Substance P produced a concentration dependent increase in short-circuit current, the relationship showing a double sigmoidal form. The non-peptide NK1 receptor antagonist, CP 99,994 ((2S,3S)-3-(2-methoxybenzyl)amino-2-phenylpiperidine), caused a dextral shift of the first sigmoidal response, indicating the involvement of an NK1 receptor. This was further supported by a concentration-dependent response of the NK1 receptor agonist [Sar9Met(O2)11]substance P with an EC50 value of 235.0+/-53.9 nM. Increasing concentrations of CP 99,994 (0.1, 0.3 and 1 microM) produced a parallel dextral shift of the [Sar9Met(O2)11]substance P curve with a slope of the Schild regression significantly different from unity (1.59). The neurokinin A concentration-response curve, with an EC50 value of 68.87+/-16.23 nM, was not significantly changed by the non-peptide NK2 receptor antagonist SR 48,968 ((S)-N-methyl-N-(4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophe nyl)butyl)bezamide). In additional studies, the peptide NK2 receptor antagonists, GR 94,800 (PhCO-Ala-Ala-DTrp-Phe-DPro-Pro-NleNH2) and PD 147,714 ((2,3-diOMeZ)-(S)Trp(S)alphaMePheGlyNH2), did not change the response to neurokinin A. However, CP 99,994 totally inhibited neurokinin A responses at 0.5 microM and above. The NK2 receptor agonist, [beta-Ala8]neurokinin A-(4-10), caused only an increase in short-circuit current in microM concentrations, whereas the NK3 receptor agonist, senktide, did not elicit a response. These results indicate, that substance P and neurokinin A mediate ion transport in porcine jejunum through NK1 receptors. However, tachykinins seem to activate another receptor. Two active conformers of the NK1 receptor might be present.

The stress of not being able to perform dustbathing in laying hens.

The stress of not being able to perform dustbathing was studied in small groups of laying hens. The birds were reared and kept for 2.5 years in cages with either sand ("sand birds") or wire floors ("wire birds"), and subsequently deprived of sand (sand birds) or given access to sand (wire birds). Before this change, wire birds had a higher incidence of unilateral wing/leg stretching and stereotypic pecking compared to sand birds; however, there was no difference in corticosterone concentrations. Deprivation of sand in the sand birds resulted in a total absence of dustbathing and in a significant increase in corticosterone concentrations. Although the wire birds dustbathed on the wire before the change of floors, there was a significant increase in the incidence of dustbathing after sand was provided, but no changes in the concentrations of corticosterone. Threats and allopecks decreased in the wire birds after access to sand, whereas no changes were found in the sand birds. Stereotypic pecks on feathers were absent in the sand birds, but were frequent in most of the wire birds both before and after they were given sand. Also, in the wire birds, all feather pecking (stereotypic and nonstereotypic) was positively correlated with the concentration of corticosterone, and this kind of pecking may, thus, be an expression of stress in laying hens. We conclude that the nonperformance of dustbathing behavior is associated with the experience of stress.

Differences in epithelial morphology correlate to Na(+)-transport: a study of the proximal, mid, and distal regions of the coprodeum from hens on high and low NaCl diet.

A study was performed to correlate regional morphology and amiloride inhibitable Na(+)-transport in the coprodeal epithelium in hens, Gallus domesticus, on low-NaCl diet and in controls. Proximal (close to colon), mid and distal (close to urodeum) regions were examined using light microscopy, transmission- and scanning electron microscopy. Na(+)-transport was measured electrophysiologically in Ussing-chambers in the proximal and distal regions. The epithelium, simple and columnar, is composed of absorptive intestinal epithelial cells, goblet cells, brush cells, migrating lymphoid cells, and enteroendocrine cells. Brush cells, identified in avians for the first time, occur in highest number in the proximal part of the coprodeum in low-NaCl hens. Na(+)-transport is high in the low-NaCl hens, ranging from 347 microA/cm2 (proximal) to 187 microA/cm2 (distal). In control hens, which correspond to hens on high-NaCl diet, it is low in all regions (0-4 microA/cm2). Absorptive intestinal epithelial cells as well as brush cells adapt to variations in transepithelial Na(+)-transport by regulating height and packing density of their microvilli, number, size, and localization of apical vesicles, and the width of the intercellular space. Regional differences in the epithelial cell composition and ultrastructure are closely correlated to transepithelial Na(+)-transport but only in low-NaCl hens, as controls do not show these variations.

Gestational profile of the stimulatory effects of porcine amniotic and allantoic fluids on prostaglandin G/H synthase activity.

The effects of porcine fetal fluids (amniotic and allantoic) on microsomal prostaglandin G/H synthase (PGHS) activity were assessed. Both amniotic and allantoic fluids obtained from late-gestation sows stimulated PGHS activity (as indicated by increased formation of radiolabelled prostaglandin) in a dose-dependent manner. At the maximum dose tested, amniotic and allantoic fluids stimulated prostaglandin (PG) formation by 55.5 +/- 1.5 and 58.5 +/- 4.7%, respectively (n = 3, P less than 0.01). Based upon ED50 values, amniotic fluid was approximately threefold more effective than allantoic fluid in stimulating PG formation. The stimulatory effect of amniotic but not allantoic fluid increased significantly (P less than 0.01) during gestation (Days 47-112). The observed changes in the stimulatory effect of amniotic fluid on microsomal PG formation parallels the in vivo changes that occur in intra-uterine PG synthesis. Amniotic fluid stimulatory activity may contribute to this gestational increase in PG synthesis.

New aspects of the pathophysiology and treatment of secretory diarrhoea.

This review presents recent findings regarding the physiological and pathophysiological extra- and intracellular mechanisms of secretory diarrhoea. Putative interventions directed towards counteracting the mechanisms causing fluid loss, especially in relation to the enteric nervous system, intracellular mediators, and localization of fluid and electrolyte transport, are discussed. The enteric nervous system regulates the complex process of transmural fluid and electrolyte transport by controlling the function of the mucosa, the motility, and the microcirculation in both health and disease. Most of the processes, leading to secretory diarrhoea, involve activation of the enteric nervous system, with local release of neurotransmitters and other endogenous effectors, which induce chloride secretion. A new therapeutic approach is based on stimulation of absorption and inhibition of secretion by using receptor agonists and antagonists, and modulators of intracellular signal transduction. A physio-pharmacological review of serotonin and the antisecretory factor as modulators of intestinal fluid and electrolyte transport is given.

Effect of indomethacin, renzapride, methysergide, ketanserin, granisetron and citalopram on serotonin-induced fluid accumulation in pig jejunum.

The purpose of this study was to elucidate the intestinal serotonin (5-HT) receptor subtypes involved in fluid transport in the pig jejunum in vivo. The fluid accumulating effect of intraluminally administered 5-HT, renzapride, methysergide, ketanserin, granisetron, citalopram and intravenous indomethacin, was tested in tied-off loops in vivo. 5-HT caused a dose-dependent fluid accumulation, which was reduced by indomethacin by about 30%. Renzapride, methysergide, ketanserin, granisetron and citalopram all caused fluid accumulation. Taking into account these fluid accumulating effects, renzapride, methysergide, ketanserin and granisetron reduced the fluid accumulating effect of 5-HT, giving a maximal reduction of 70, 46, 76, and 80%, respectively. These data suggest the existence of intestinal 5-HT receptor subtypes involved in fluid transport in the pig jejunum. The antagonistic effects of indomethacin, ketanserin and granisetron, suggest the involvement of prostaglandins, as well as the 5-HT2 and the 5-HT3 receptor subtypes in the fluid accumulating response of 5-HT.

Water absorption in relation to fermentation in the colon of the ostrich (Struthio camelus).

The colon is a major site for fermentation and water absorption in the ostrich. Water absorption along the colon was evaluated and its relationship to osmolality, Na+ concentration, short chain fatty acid (SCFA) concentration and carbohydrate content of digesta analysed. Mean water content decreased from 5.30 +/- 0.99 to 2.51 +/- 0.13 mf/g dry mass in the first 5 m of the colon. Correspondingly, mean carbohydrate content fell from 529.85 +/- 46.61 to 434.99 +/- 29.89 mg/g dry mass. A significant correlation was shown between the decreases in mean carbohydrate and water content along the colon (r2 = 0.997, P < 0.05). Changes in mean osmolality (+/- 10 mOsm/kg) and SCFA concentration (+/- 7 mmol/l) were minimal in comparison to the change in Na+ concentration (-54 mmol/l). These findings reflect a close coupling between SCFA production and absorption on the one hand and water absorption on the other.

Experimental studies of intestinal ion and water transport.

A major advance in transport physiology was H. H. Ussing's development of the voltage-clamp method, and later the Koefoed-Johnsen-Ussing model for Na+ transport. In the same decade, J. C. Skou identified the Na(+)-K(+)-ATPase, which maintains the Na+ and K+ gradients that drive most epithelial transport processes. With this foundation, Danish scientists have pursued the mechanism of ion transport and the resulting solute-linked water flow. Recent contributions have been on isosmotic transport, suggesting solute recycling, and KCl-water cotransport in the basolateral epithelial cell membrane. Efficient small intestinal nutrient absorption is dependent on coupling to the Na+ gradient. Cotransport of Na+ and glucose is quantitatively the most important absorptive mechanism in the small intestine, as illustrated by the success of oral rehydration solutions in diarrhoea. The majority of amino acids are likewise transported by Na+ dependent carriers, but recent experiments have identified a concomitant Cl- dependency for some. Regulation of intestinal secretion, both under normal digestive processes, and in response to enterotoxins, has turned out to be very complex. It involves local and central neuronal regulation through an array of neurotransmitters and local actions of gastrointestinal hormones. Major effectors are the submucosal neurons and the main transmitters serotonin, vasoactive intestinal peptide, acetylcholine, substance P, and neurotensin. Development of antisecretagogues is impeded by the existence of several receptor subtypes and significant species differences. The Na+ and water-conserving properties of the large intestine have been shown to be regulated by adrenocortical hormones, with aldosterone as a potent stimulator of colonic Na+ absorption. A major colonic function is the symbiosis with the anaerobic bacterial population. The fermentation of carbohydrate to short-chain fatty acids, which can be absorbed, supplements small intestinal digestive function.

The mechanism of salt and water absorption in the intestine of the eel (Anguilla anguilla) adapted to waters of various salinities.

1. The absorption of NaCl and water was studied by intraluminal in vivo perfusion of the intestine of the yellow European eel (Anguilla anguilla) adapted to fresh water (FW), to sea water (SW), and to double strength SW (DSW).2. The net lumen to plasma NaCl transport from diluted SW perfusion fluids was independent of the NaCl concentration in the Na(+) concentration range tested. The NaCl absorption (expressed as mu-equiv/100 g.hr.) increased from FW (mean +/- S.E.): Na(+) 166 +/- 17, Cl(-) 205 +/- 24 to SW: Na(+) 363 +/- 33, Cl(-) 423 +/- 37, and again in DSW: Na(+) 640 +/- 110, Cl(-) 676 +/- 149.3. The osmolality of the perfusion fluid which resulted in zero net water transport was higher than plasma osmolality by 73 +/- 3 m-osmole in FW, 126 +/- 5 m-osmole in SW, and 244 +/- 32 m-osmole in DSW (mean +/- S.E.). A fairly constant ratio between net NaCl transport and this osmolality difference prevailed.4. The general osmotic permeability to water in the serosa-mucosa direction (expressed as mul./100 g. hr. m-osmole) measured from experiments with impermeant solute increased from FW: 3.7 +/- 0.5 to SW: 7.2 +/- 1.0 (mean +/- S.E.).5. These results are compatible with the interpretation that the water flow occurring in the absence of a general transmural osmotic gradient, the ;solute-linked water flow', is linearly related both to net NaCl transport and to the osmotic permeability to water. The findings support the view that the ;solute-linked water flow' is, indeed, secondary to the salt movement and is due to osmotic force.6. The amount of water absorbed from dilute SW perfusion fluids isosmotic with plasma was larger than in most other intestinal epithelia. FW: 650, SW: 1620 mul./100 g. hr. The NaCl concentration of the absorbate was hypertonic to plasma.7. The passive permeability of the intestine to NaCl was very low, and the reflexion coefficient was close to unity. Therefore metabolic energy will be used to absorb NaCl, even when the NaCl concentration in the gut is higher than that of plasma due to ingestion of SW. There appears to be a limited interaction in the intestinal wall between passive salt and water flow.8. In DSW the Na(+) ingestion with the oral intake of the surrounding fluid matched the gut absorption capacity. Since DSW is close to the tolerance limit, it is concluded that the gut NaCl transport capacity may be one of the factors limiting the tolerance to water or higher salinity.