Regulatory role of cAMP in transport of Na+, Cl- and short-chain fatty acids across sheep ruminal epithelium

Sodium is absorbed in considerable amounts across the ruminal epithelium, whilst its transport is strongly interrelated with the permeation of chloride and short-chain fatty acids (SCFAs). However, regulation of ruminal Na+, Cl-, and SCFA absorption is hardly understood. The present study was therefore performed to characterize the influence of cAMP on sodium and sodium-coupled transport mechanisms in short-circuited, stripped ruminal epithelia of sheep. Elevation of intracellular cAMP concentrations by theophylline (10 mM) or theophylline in combination with forskolin (0.1 mM) significantly reduced mucosal-to-serosal sodium transport, leading to a reduction of net transport. The theophylline- or theophylline-forskolin-induced reduction of sodium transport was accompanied by a decrease in chloride net transport but revealed no effect on propionate flux. Short-chain fatty acids stimulated Na+ transport but their stimulatory effect was almost completely blocked by theophylline-forskolin. In solutions with and without SCFAs, the inhibitory effect of 1 mM amiloride on sodium transport was strongly reduced after theophylline-forskolin pretreatment of the tissues. Blocking the production of endogenous prostaglandins by addition of indomethacin (10 microM) led to a theophylline-sensitive stimulation of unidirectional and net fluxes of sodium. The findings indicate that apical, amiloride-sensitive Na+-H+ exchange and/or basolateral Na+-K+-ATPase can effectively be blocked by cAMP, leading to a decrease in sodium and chloride transport. In the ruminal epithelium, cAMP is a second messenger of prostaglandins, which are released spontaneously under in vitro conditions.     

The role of volume-sensitive CI channels in the stimulation of chloride absorption by short-chain fatty acids in the rat colon

The short-chain fatty acids acetate, propionate and butyrate induced a concentration-dependent decrease in short-circuit current (I_sc) of the rat colon in vitro. The decrease in I_sc, being more pronounced in the distal than in the proximal colon, was dependent on the presence of CI^- ions and partly on the presence of HCO^-₃. In the distal colon, the fall in I_sc could be inhibited by amiloride, indicating that the activity of the Na⁺/H⁺ exchanger is necessary for the induction of this response. The decrease in I_sc was diminished by the CI^- channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate, and the lipoxygenase inhibitor, nordihydroguaiaretic acid. In contrast, inhibitors of the leukotriene pathway or a CI^- channel blocker did not affect the I_sc response in the proximal colon. Measurements of unidirectional fluxes revealed that butyrate caused a stimulation of the mucosa to serosa fluxes (J_ms) of Na⁺ and CI^- in the distal, but only of j_ms^Na in the proximal colon. Unidirectional Rb⁺ fluxes were not altered. The stimulation of j_ms^cl correlated with the degree of metabolism of the short-chain fatty acid. The increase in j_ms^cl was most pronounced for butyrate, smaller for acetate and not observed with the poorly metabolizable short-chain fatty acid, isobutyrate. Consequently, two factors seem to be responsible for the stimulation of CI^- absorption by short-chain fatty acids in the distal colon: (1) the intracellular production of HCO^-₃ during the oxidation of short-chain fatty acids as substrate for the apical Cr/HCO^-₃ exchanger, and (2) the activation of volume-sensitive basolateral CI^- channels.     

Segmental differences in electrical properties and Na-transport of rabbit caecum,proximal and distal colon in vitro

Electrical potential difference, short circuit current, tissue conductance, and unidirectional Na-fluxes were measured in four segments of the rabbit large intestine in vitro. Compared to the relatively tight, low conductance distal colon, caecum and proximal parts of the colon are leaky epithelia with high conductances. Net Na-absorption was highest in caecum, and then decreased gradually towards the distal colon, whereas potential and short circuit current where high in the caecum and proximal colon, low in the middle part of the colon, and high again in the distal colon. Unidirectional Na-fluxes of all four segments were different.The discrepancy between the short circuit current and net Na-absorption in the two segments of the proximal colon indicates electrogenic transport of other ions. 0.1 mM ouabain virtually abolished short circuit current and Na-absorption in all segments, whereas 0.1 mM amiloride was not effective in the caecum and the proximal colonic parts. The present study focuses on the comparative aspects of Na-transport. It demonstrates the marked segmental heterogeneity of the basic electrical properties and suggests four different segmental organizations of large intestinal electrolyte transport.     

Short-chain fatty acids decrease the frequency of spontaneous contractions of longitudinal muscle via enteric nerves in rat distal colon

Short-chain fatty acids (SCFAs) produced by the bacterial fermentation of carbohydrates in the cecum and proximal colon are reported to modify colonic motility as a luminal factor. Besides the physical stimuli in the distal colon, SCFAs in the intestinal lumen also seem to affect colonic motility under physiological concentrations. This study therefore used fasted rats to investigate the effect of SCFAs on the spontaneous contractions of longitudinal muscle (LM) in rat distal colon, including mucosa in vitro. The frequency of spontaneous contractions of LM strips from the distal colon was 9.4 +/- 0.5 contractions/20 min. The exogenous addition of >5 mM SCFAs decreased the frequency of spontaneous contractions of the LM to 6.1 +/- 0.8 contractions/20 min. Among SCFAs, only acetate elicited this inhibitory response. TTX and the combination of hexamethonium and granisetron abolished SCFA-induced inhibitory response, suggesting that this inhibitory response is mediated via the ENS, including nicotinic and 5-HT(3) receptors. In conclusion, it is suggested that SCFAs in rat distal colon decrease the frequency of spontaneous contractions of the LM and that SCFAs may contribute to colonic motility, including the peristaltic reflex, by regulating the frequency of spontaneous contractions of the LM through the enteric nervous system (ENS).     

New clinical and experimental aspects of intestinal magnesium transport

Mg transport across various segments of the rat small and large bowel was measured in the absence of electrochemical gradients and using the voltage clamp technique. In the mucosa-to-serosa (ms) Mg flux across the duodenum, ileum, and colon a cellular part is involved, amounting to 40-70% of the total ms Mg flux measured across the short-circuited tissue. However, serosa-to-mucosa (sm) Mg flux is purely passive, suggesting that net Mg transport is largely determined by convectively driven Mg flux across the paracellular pathway. Mg is absorbed across the colon and ileum but in the duodenum paracellularly it is secreted due to an 'anomalous solvent drag effect'. Mg in the caecum decreases cellular ms Ca transport but in the other segments only passive ms Ca flux is reduced by a decrease of the paracellular permeability for Ca, or by a Mg-induced decrease in water absorption. Mg transport across all segments is insensitive to 1,25(OH)2D3. Dexamethasone abolishes cellular Ca transport but stimulates paracellular ms Mg flux in the duodenum. It is concluded that in the rat (a) Mg and Ca are transported by distinct cellular and paracellular mechanisms; (b) Mg transport is largely confined to the paracellular pathway; (c) the ileum and the colon are the major sites for the gut regulation of Mg homeostasis.     

Adaptation of electrolyte transport in rat large intestine after proximal resection

Electrolyte transport was studied in rat colon adapting to 50% small intestinal resection with cecectomy. Four weeks after surgery, colonic gross surface area, dry and wet weight were increased compared to sham-operated controls. Net absorption in vivo of sodium, chloride and volume was stimulated per organ and per unit tissue mass, and net potassium secretion was diminished. The electrical potential difference, mucosal Na-K-ATPase specific activity and cAMP concentration remained unaffected. In vitro, measurements of unidirectional fluxes and electrical parameters across isolated mucosa indicated enhanced electrically neutral sodium chloride absorption in the proximal and possibly diminished bicarbonate secretion in the distal colon. Thus, removal of the cecum in addition to partial jejunoilectomy induces profound adaptive changes in the colon, involving not only mucosal growth but also a functional reaction of the individual epithelial cell.     

The protein tyrosine kinase pathway is not involved in the regulation of K+ transport across the rat colon.

The protein tyrosine kinase inhibitor, genistein, is known to activate the cystic fibrosis transmembrane regulator (CFTR) Cl- channel and to inhibit K+ currents across the rat colonic epithelium. The aim of the present study is to answer the question whether these effects are involved in the regulation of transepithelial K+ transport. Therefore, the action of genistein on K+ transport in rat proximal and distal colon was studied by measuring unidirectional fluxes, uptake and efflux of Rb+ in mucosa-submucosa preparations. All effects of genistein (5 x 10(-5) mol L(-1)) were tested in the presence of a low concentration of forskolin (2 x 10(-7) mol L(-1)), because prestimulation of the cAMP pathway has been shown to be a prerequisite for a secretory action of genistein. Forskolin caused an increase in the serosa-to-mucosa flux of Rb+ (J(Rb)sm) thereby stimulating net K+ secretion in the proximal and distal colon. None of these effects was further enhanced after administration of genistein. Neither mucosal uptake of Rb+, representing mainly the activity of the H+-K+-ATPase in the distal colon, nor serosal Rb+ uptake, representing, e.g. the activity of the Na+-K+-2Cl- cotransporter, were affected by genistein. Also the efflux of Rb+ across the apical or the basolateral membrane, an indicator for the apical and basolateral K+ conductance, was unchanged in the presence of genistein. These results demonstrate that the K+ channels inhibited by genistein are not involved in transepithelial K+ transport.     

Transport of electrolytes across the helicoidal colon of the new-born pig.

1. The Na, K, Cl and water content of faeces removed from different parts of the pig helicoidal colon were determined for 1-day-old and adult animals. Faecal Na, Cl and water content fell in both cases during passage of contents through the colon. K content increased in the distal colon of the adult pig. This did not occur in the 1-day-old animal. 2. The colon of the 1-day-old pig removed a larger proportion of water from its contents than did that of the adult. The absorption of both water and Cl was found to extend into the distal colon of the 1-day-old animal; little or no net absorption took place in this region in the adult. 3. Colons taken from new-born pigs maintained stable short-circuit currents of about 60-80 muA cm-2 with open-circuit voltages of about 10 mV. Similar values were found for proximal and mid regions of colons taken from 1-day-old, suckled animals. In the distal colon, however, both short-circuit current and open-circuit potential doubled after suckling. 4. Measurements of Na flux in vitro showed no regional difference at birth. The amount of Na absorbed, about 4.5 muequiv cm-2 h-1, was twice that predicted from the short-circuit current, supposing that to be due solely to the electrogenic transport of Na. 5. Colons taken from suckled pigs transported Na at double the rate found in the new-born animal. This applied to both the proximal region, where the short-circuit current remained constant, and the distal region, where the short-circuit was double that of the new-born. 6. Fluxes of Cl and K were also measured across the proximal colon of the one-day-old, suckled pig. There was a net absorption of Cl and secretion of K (1.3 and -0.05 muequiv cm-2 h-1 respectively). These fluxes, taken together with that for Na, could not wholly account for the short-circuit current measured across these preparations. 7. The pig colon seems well able to cope with both electrolyte and water absorption during the first 24 hr of post-natal life. Part of the absorbed sodium appears to follow a non-electrogenic, possibly pinocytotic, route, but the full ionic contribution to the measured short circuit current has still to be determined.     

Intestinal lipid metabolism in suckling rats infected withGiardia duodenalis

We carried out a quantitative and qualitative analysis of intestinal digestion of neutral lipids in suckling rats infected withGiardia duodenalis. Total lipids were measured after extraction from the contents of the stomach, proximal and distal small bowel, caecum and colon. Amounts gradually fell from the stomach to the colon and were identical in infected animals and controls, although high values were occasionally found in the caecum of infected rats. Relative glyceride quantities were determined by means of high-performance thin-layer chromatography. Triglycerides were absent from the distal small bowel, and only free fatty acids and cholesterol were present in the caecum, reflecting normal digestion of neutral lipids in infected suckling rats. Our results suggest thatG. duodenalis does not impair intestinal fat digestion in suckling rats.     

Effects of nicotinic receptor blockade on the colonic mucosal response to luminal bile acids in anaesthetized rats

AIM: To test the hypothesis that nicotinic receptor mechanisms mediate the effects of bile acids on the colonic mucosa. METHODS: The epithelial transport response to 4 mm deoxycholic acid (DCA) was studied in vitro and in vivo, in rat colon. In vitro, epithelial resistance was measured by square pulse analysis, and net membrane current was calculated from the transmucosal potential difference (PD) and resistance. In vivo, we measured PD and net fluid transport. RESULTS: In vitro, DCA significantly increased membrane current and induced a progressive decrease in epithelial resistance, which in the distal colon eventually resulted in a significant PD reduction. This response was not significantly affected by hexamethonium. In vivo, DCA reduced PD with a significantly larger response in distal colon, but had no consistent effect on net fluid absorption. Nicotinic receptor blockade per se increased net fluid absorption and slightly reduced PD in proximal colon, and inhibited spontaneous net fluid secretion and markedly reduced PD in distal colon. Nicotinic receptor blockade significantly attenuated the bile-acid induced PD response. CONCLUSION: The data do not support the theory that a bile acid-activated secretory reflex exists in rat colon. The reduced PD response after hexamethonium suggests that a mechanism involving nicotinic receptors may potentiate the permeability response to luminal bile acids.     

Monocarboxylate transporter 1 (MCT1) plays a direct role in short-chain fatty acids absorption in caprine rumen

Despite the importance of short-chain fatty acids (SCFA) in maintaining the ruminant physiology, the mechanism of SCFA absorption is still not fully studied. The goal of this study was to elucidate the possible involvement of monocarboxylate transporter 1 (MCT1) in the mechanism of SCFA transport in the caprine rumen, and to delineate the precise cellular localization and the level of MCT1 protein along the entire caprine gastrointestinal tract. RT-PCR revealed the presence of mRNA encoding for MCT1 in all regions of the caprine gastrointestinal tract. Quantitative Western blot analysis showed that the level of MCT1 protein was in the order of rumen ≥ reticulum > omasum > caecum > proximal colon > distal colon > abomasum > small intestine. Immunohistochemistry and immunofluorescence confocal analyses revealed widespread immunoreactive positivities for MCT1 in the caprine stomach and large intestine. Amongst the stratified squamous epithelial cells of the forestomach, MCT1 was predominantly expressed on the cell boundaries of the stratum basale and stratum spinosum. Double-immunofluorescence confocal laser-scanning microscopy confirmed the co-localization of MCT1 with its ancillary protein, CD147 in the caprine gastrointestinal tract. In vivo and in vitro functional studies, under the influence of the MCT1 inhibitors, p -chloromercuribenzoate (pCMB) and p -chloromercuribenzoic acid (pCMBA), demonstrated significant inhibitory effect on acetate and propionate transport in the rumen. This study provides evidence, for the first time in ruminants, that MCT1 has a direct role in the transepithelial transport and efflux of the SCFA across the stratum spinosum and stratum basale of the forestomach toward the blood side.     

Effect of short-chain fatty acids on calcium absorption by the rat colon

An in vivo luminal perfusion technique was used to investigate whether short-chain fatty acids influence the absorption of Ca by the rat colon. Na and water absorption were also determined. In the distal colon, acetate and butyrate caused a significant increase in Ca absorption, while the absorption of Na and water were not affected. In the proximal colon, butyrate did not influence Ca absorption, but significantly enhanced Na and water absorption. These results are in part consistent with the presence of a Ca-H exchanger in the apical membrane of the distal colon mediating Ca uptake into the epithelial cell.     

Intracellular pH regulation in guinea-pig caecal and colonic enterocytes during and after loading with short-chain fatty acids and ammonia

Absorption of short-chain fatty acids (SCFA) and ammonia implies considerable fluxes of protons across the epithelium of the large intestine. Efficient regulation of intracellular pH (pH(i)) is therefore essential in these cells. The aim of the present study was to examine the effects of SCFA and of ammonia on pH(i), on pH(i) regulation and to characterize the mechanisms involved in pH(i) regulation in surface enterocytes of the guinea-pig caecal and colonic mucosa. Intact epithelia from the caecum and the distal colon were mounted in a microperfusion chamber. pH(i) was measured by fluorescence microscopy using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Addition of SCFA or ammonia to the serosal side changed the enterocyte pH(i) markedly, whereby ammonia caused larger changes in pH(i) than SCFA. In contrast, addition of SCFA to the mucosal solution had no effect on pH(i) and ammonia increased pH(i) only slightly. Basolaterally located pH(i) regulation mechanisms, Na(+)-H(+) exchange and Cl(-)-HCO(3)(-) exchange, are involved mainly in returning pH(i) to normal values. It is concluded that, due to apparently lower permeability of the apical membranes, the caecal and colonic epithelium is protected against pH(i) disturbances caused by the naturally high luminal SCFA and NH(3) concentrations. The major regulation mechanisms of pH(i) are located in the basolateral membrane of the enterocytes.     

Effects of trifluoperazine, ouabain, and ethacrynic acid on intestinal calcium transport

Bidirectional steady-state calcium fluxes were measured in vitro under short-circuited conditions in segments of rat duodenum and descending colon. The calcium-activated ATPase (Ca-ATPase) inhibitor trifluoperazine (TFP, 0.1 mM) reduced net calcium absorption in both tissues by decreasing the absorptive flux from mucosa to serosa (Jm leads to s) without consistently altering the secretory flux from serosa to mucosa. 1,25-Dihydroxyvitamin D3 administration (50 ng/day for 4 days) increased net calcium absorption by increasing Jm leads to s, and TFP reduced Jm leads to s to the same extent across tissues from vehicle- or 1,25-dihydroxyvitamin D3-treated animals. Na-K-ATPase inhibitors ouabain and ethacrynic acid both reduced short-circuit current without affecting calcium fluxes. These data suggest that Ca-ATPase, located in the basolateral membrane of intestinal epithelial cells, plays a role in the transepithelial transport of calcium. More general effects of TFP on intestinal epithelium may also contribute to the reduction in calcium fluxes. Duodenal and descending colon calcium transport appears independent of transcellular sodium transport mediated by Na-K-ATPase.     

Influence of short-chain fatty acids on ammonia absorption across the rumen wall in sheep.

The effects of short-chain fatty acids (SCFA) on ammonia net absorption from the sheep rumen in vivo and on ammonia transport across rumen wall mucosa in vitro were studied. Ammonia net absorption was directly, though in a non-linear manner, correlated with the SCFA concentration in the artificial rumen fluid. Almost 70% of total ammonia absorption was dependent upon the presence of SCFA when 12 mmol l-1 ammonia and 67.5 mmol l-1 SCFA were present. Lactic acid was ineffective. Incubation experiments showed that mucosal disappearance and serosal appearance of ammonia were reduced by 38% and 32%, respectively, when SCFA (63 mmol l-1) were replaced by lactic acid. The SCFA effect was independent of the type of SCFA used. In part of the experiments up to 54% of the ammonia taken up by the tissue was not recovered in the serosal incubation solution and must have been metabolized in the mucosa.     

Too much short chain fatty acids cause neonatal necrotizing enterocolitis

Nenatal necrotizing enterocolitis (NEC) is a disease mainly affects premature infants. It is well known that prematurity, enteral formula feeding, and bacterial colonization are three major risk factors for NEC. Acetic acid, propionic acid and butyric acid are short chain fatty acids (SCFAs), which are produced mainly in the colon by bacterial fermentation of undigested carbohydrates. Although luminal production of modest quantities of SCFAs is essential for normal colonic mucosal function, excessive production/accumulation of SCFAs may arise in premature infants due to increased luminal carbohydrates malabsorption and poor gastrointestinal motility, and may have deleterious effects on mucosal integrity. Therefore, it is proposed that too much luminal short chain fatty acids cause neonatal NEC.     

Hexose transport across the apical and basolateral membrane of enterocytes from different regions of the chicken intestine

The properties of hexose transport across the apical and basolateral membranes of chicken enterocytes have been studied in the small and large intestine. Results show that (a) isolated epithelial cells from all segments except the coprodeum can accumulate 3-O-methylglucose (Glc3Me) against a concentration gradient, by a Na⁺-dependent and phloridzin-sensitive mechanism, (b) The cell cumulative capacity for Glc3Me (control/phloridzin-incubated cells) is lower in the small intestine than in the large intestine (rectum = proximal caecum = ileum > jejunum > duodenum). (c) Theophylline enhances the cell Glc3Me cumulative capacity 2.9-fold in the duodenum and 2.4-fold in the jejunum but has no effect in the other segments studied. (d) Analysis of sugar uptake indicates that net hexose influx rates decrease from proximal to distal regions: jejunum > duodenum > ileum = proximal caecum = rectum for the apical transport system (-methyl glucoside as substrate and phloridzin as inhibitor) and duodenum > jejunum > ileum = proximal caecum = rectum for the basolateral system (2-deoxyglucose; theophylline). (e) The duodenum and the jejunum show high apical and basolateral hexose transport rates, which confer a significant capacity for sugar absorption on the proximal intestine. More distal regions, including the ileum, the proximal caecum and the rectum, have transport systems analogous to those of the proximal intestine that keep a considerable potential capability to recover hexoses from the lumen.     

Morphology and immunoperoxidase studies of intestinal adenomatosis in the blue fox, Alopex lagopus

In a blue fox farm about 300 of 400 weanling pups were affected with diarrhoea and prolapse of the rectum. About 40 pups died in an emaciated state after a few days of disease. Eight pups were submitted for examination. Pathological findings were restricted to the caecum, proximal colon and rectum, with occasional extension to the ileum. Affected areas revealed a thick and rigid intestinal wall, a narrow lumen and a thickened, wrinkled and sometimes ulcerated mucosa. Histologically, the mucosa showed long tortuous crypts outlined by a high pseudostratified epithelium which lacked goblet cells. Epithelial cells revealed increased basophilia, and mitoses were seen along the entire crypt length and in the surface epithelium. Silver impregnation showed curved organisms in the apical cytoplasm of the altered epithelial cells. Penetration of the muscularis mucosa by the altered epithelial cells was frequent. The avidin-biotin-immunoperoxidase complex (ABC) technique revealed positive organisms in the apical cytoplasm of altered epithelial cells, when rabbit antisera against different biotypes and serotypes of Campylobacter jejuni and Campylobacter coli were used. Antisera against Campylobacter sputorum subsp. muscosalis and Campylobacter hyointestinalis were negative.     

The site and characteristics of intestinal water and electrolyte loss in Escherichia coli-induced diarrhoea in calves

Diarrhoea was induced in neonatal calves by the oral administration of an enteropathogenic strain of Escherichia coli. The calves were protected by IgM against septicaemia. Under anaesthesia, movements of water and electrolytes were measured at four points in the small intestine and at one point in the proximal spiral colon. The results were compared with those from healthy control animals.Control animals showed net secretion of sodium and chloride in the upper jejunum changing progressively to net absorption in the distal ileum. Bicarbonate was absorbed in the proximal and secreted in the distal small intestine, and potassium was secreted at all points examined.In the diarrhoeic animals there were increased net losses of water, sodium, potassium and chloride in the distal small intestine, whereas the spiral colon of these animals showed significantly greater net absorption of water and sodium than did the controls. However, potassium losses in the colon were greater in the diarrhoeic calves. No significant difference in net bicarbonate movement could be shown between the two groups.Unidirectional fluxes of water and sodium were significantly reduced throughout the intestine in the diarrhoeic animals compared with the controls.It was concluded that the distal small intestine was the main site of net loss of water and electrolytes, but that there was partial compensation by the proximal spiral colon for losses of water and sodium. This may have been mediated by aldosterone.