Segmental action of aldosterone on water and electrolyte transport across rabbit colon in vivo

Water and electrolyte transport (Na, K, Cl) and the electrical potential difference were investigated simultaneously in five segments of rabbit colon in vivo, under control conditions and under the influence of aldosterone. A dialysis method was used. The solution was designed to mimic the electrolyte concentration of the lumen, and to simulate normal in vivo conditions. Under in vivo conditions (imposed gradients), marked segmental differences in water and electrolyte transport were present. The proximal colon secreted sodium and absorbed potassium, whereas the distal colon absorbed sodium and secreted potassium. Chloride was secreted in all segments. Aldosterone stimulated water and NaCl absorption in the proximal colon by an electroneutral mechanism, whereas in the distal colon a stimulation of electrogenic Na-absorption occurred. The results demonstrate the role of aldosterone in regulating rabbit colonic electrolyte transport. A uniform hormonal stimulation causes qualitatively and quantitatively different effects in the various segments.     

Opioid receptor agonists in the rabbit colon: comparison of in vivo and in vitro studies

Myoelectrical activity was recorded in the proximal and distal colon of rabbits using chronically implanted electrodes. The motility in both the proximal and distal colon was inhibited by the intravenous (IV) administration of the following opioid agonists for mu receptors: morphine and fentanyl, kappa receptors: ethylketazocine (EKC) and U 50 488 H, and delta receptors: D-Ala2 D-Leu5-enkephalin (DADLE) and D-Ser2 Leu-enkephalin-Thr6 (DSLET). In contrast, the myoelectric activity in the distal colon was increased during the infusion of an endogenous kappa opioid agonist, dynorphin (DYN). All of these effects were prevented by naloxone pretreatment. During in vitro studies using extraluminal force transducers, fentanyl, U 50 488 H and DSLET inhibited spontaneous contractions of the proximal colon, but U 50 488 H and DSLET caused a substantial increase in the motility of the distal colon. The observed motor responses in the proximal and distal colon following opioid agonist administration indicate that the control of these two intestinal segments may be different. It is suggested that the stimulatory effect of dynorphin on the distal colon is peripherally-mediated while inhibition of the whole colon by opioid agonists regardless of subtypes seems to be centrally-mediated.     

Effect of E. coli heat-stable enterotoxin on colonic transport in guanylyl cyclase C receptor-deficient mice

We studied the functional importance of the colonic guanylyl cyclase C (GCC) receptor in GCC receptor-deficient mice. Mice were anesthetized with pentobarbital sodium, and colon segments were studied in Ussing chambers in HCO3- Ringer under short-circuit conditions. Receptor-deficient mouse proximal colon exhibited similar net Na+ absorption, lower net Cl- absorption, and a negative residual ion flux (J(R)), indicating net HCO3- absorption compared with that in normal mice. In normal mouse proximal colon, mucosal addition of 50 nM Escherichia coli heat-stable enterotoxin (STa) increased the serosal-to-mucosal flux of Cl- (J(s-->m)(Cl)) and decreased net Cl- flux (J(net)(Cl)) accompanied by increases in short-circuit current (I(sc)), potential difference (PD), and tissue conductance (G). Serosal STa had no effect. In distal colon neither mucosal nor serosal STa affected ion transport. In receptor-deficient mice, neither mucosal nor serosal 500 nM STa affected electrolyte transport in proximal or distal colon. In these mice, 1 mM 8-bromo-cGMP produced changes in proximal colon J(s-->m)(Cl) and J(net)(Cl), I(sc), PD, G, and J(R) similar to mucosal STa addition in normal mice. We conclude that the GCC receptor is necessary in the mouse proximal colon for a secretory response to mucosal STa.     

Intestinal permeability and contractility in murine colitis.

We developed an in vitro organ bath method to measure permeability and contractility simultaneously in murine intestinal segments. To investigate whether permeability and contractility are correlated and influenced by mucosal damage owing to inflammation, BALB/c mice were exposed to a 10% dextran sulphate sodium (DSS) solution for 8 days to induce colitis. The effect of pharmacologically induced smooth muscle relaxation and contraction on permeability was tested in vitro. Regional permeability differences were observed in both control and 10% DSS-treated mice. Distal colon segments were less permeable to 3H-mannitol and 14C-PEG 400 molecules compared with proximal colon and ileum. Intestinal permeability in control vs. 10% DSS mice was not altered, although histologic inflammation score and IFN-gamma pro-inflammatory cytokine levels were significantly increased in proximal and distal colon. IL-1beta levels were enhanced in these proximal and distal segments, but not significantly different from controls. Any effect of pharmacologically induced contractility on intestinal permeability could not be observed. In conclusion, intestinal permeability and contractility are not correlated in this model of experimentally induced colitis in mice. Although simultaneous measurement in a physiological set-up is possible, this method has to be further validated.     

Segmental differences in short-chain fatty acid transport in rabbit colon: Effect of pH and Na

Short-chain fatty acids (SCFAs) are the predominant luminal anion in the mammalian colon. Although they are rapidly absorbed in vivo, little is known about the mechanisms of transepithelial transport in vitro. Previous studies have suggested that SCFA transport may be linked to Na absorption or an anion exchange mechanism. We compared the transport of propionate under short-circuit conditions in rabbit proximal and distal colon to determine whether there were segmental differences, how SCFAs may be linked to either Na absorption or anion transport, and whether SCFAs, as weak electrolytes, may be affected by transepithelial pH gradients. In distal colon, propionate transport was not significantly altered by stimulation of electrogenic Na absorption, epinephrine or Cl removal. However, a modest transepithelial pH gradient (luminal 6.8/serosal 7.4) stimulated propionate absorption. In proximal colon, propionate transport was significantly altered by manuevers that either stimulated (lowered [Na] in the bathing media) or inhibited (theophylline) apical Na−H exchange. Neither Cl removal, nor the anion exchange inhibitor DIDS, nor a transepithelial bicarbonate gradient, altered propionate transport. A transepithelial pH gradient inhibited propionate secretion, but not in a manner entirely consistent with the effect of pH on the distribution of a weak electrolyte. These results suggest that there is significant segmental heterogeneity in colonic SCFA transport; that transepithelial propionate fluxes are altered by changes in pH or electroneutral Na absorption (Na−H exchange), but not by chloride removal, bicarbonate gradients or electrogenic Na absorption. Regulation of SCFA transport may be an important factor in the physiology of colonic fluid balance.     

Influence of electrical gradient on electrolyte and water transport in rabbit ileum and colon in vitro

We studied the influence of the electrical gradient between the mucous and serous side on water and electrolyte transport in the rabbit distal ileum and proximal colon "in vitro". In the absence of a gradient, the absorption of water, sodium, chloride and bicarbonate rose in both ileum and colon, whereas potassium secretion tended to give way to potassium absorption.     

Electrolyte transport across rabbit late proximal colon in vitro

The second part of rabbit proximal colon was investigated in vitro under short circuit conditions. Unidirectional sodium and chloride fluxes were measured during the soft faeces period and during the hard faeces period. Rabbit late proximal colon has a potential difference (psi mS) of 4 mV, a tissue conductance (GT) of 10-11 mS/cm2 and a short circuit current (Isc) of 1.5 mueq/cm2 X hr. Under control conditions sodium (2.65 mueq/cm2 X hr) and chloride (0.67 mueq/cm2 X hr) are absorbed. Ouabain abolished psi ms,Isc and the net sodium flux totally, whereas 0.1 mM amiloride only slightly decreased the net sodium flux. No differences in electrical properties and Na,Cl-fluxes were found between the faeces periods. Removal of sodium abolished psi ms and Isc totally, and a high potassium solution depolarized the preparation (psi ms = 0). A linear current-voltage relation characterizes the tissue as an ohmic resistor between -40 and +50 mV, and reveals a slope conductance of 14 mS/cm2 under KCl conditions. We conclude that the transport functions under in vitro conditions differ markedly from the in vivo situation, and that the diurnal differences of electrolyte transport in vivo occur mainly by the involvement of ionic gradients.     

Concentration- and pH-dependence of short-chain fatty acid absorption in the proximal and distal colon of guinea pig (Cavia porcellus)

1. Absorption of short-chain fatty acids (SCFA), acetate, propionate, and butyrate was studied in simultaneously perfused proximal and distal segments of the colon in anaesthetized guinea pigs. 2. Acetate absorption rates increased linearly with concentration in both segments, indicating passive transport. 3. SCFA-clearance was independent of bulk luminal pH between pH 6.2 and 8.1 in the proximal and distal colon. SCFA-clearance was slightly higher in both segments at pH values less than 6. 4. The unexpected pH-independence of SCFA-absorption is attributed to the existence of a constant pH-microclimate at the surface of the colonic epithelium. 5. Relative permeabilities to acetate:propionate:butyrate were estimated as 1:1.19 +/- 0.03:1.27 +/- 0.05 in the proximal colon and 1:2.31 +/- 0.39:3.50 +/- 0.61 in the distal colon. The significance of these findings with respect to the pH-partition hypothesis are discussed.     

Effect of avian neurotensin on motility of chicken (Gallus domesticus) lower gut in vivo and in vitro

Mammalian neurotensin, originally isolated from bovine hypothalamus, differs from avian neurotensin (aNT) by 6 amino acid residues. Bovine neurotensin has been shown to affect motility of chicken crop and rectum and secretion of chicken ileum, but there have been no studies of the effects of aNT on avian intestinal function. This study was designed to characterize the effects of aNT on the motility of the chicken lower gut. Strain gauge transducers were used in vivo to measure contractions of chicken distal ileum, cecum, and distal colon in response to 30-min infusions of aNT at rates of 15, 30, 60 or 600 pmol.kg-1.min-1. In vitro experiments were conducted using segments of distal ileum, cecum or distal colon, stripped of mucosa, cut in either the longitudinal or circular plane, and suspended isometrically in isolated organ tissue baths at a resting tension of 1 g. Avian neurotensin, substance P (SP), or carbamylcholine (CCH) were administered to the bath and the tension generated by each tissue was recorded via a force transducer. A relaxation of chicken ileum was observed in response to aNT infusion in vivo. Except for stimulation of excretation, colon and cecum were not affected by aNT infusion. Both aNT and SP stimulated motility of chicken ileum and cecum in vitro. SP had no consistent effect on colon and aNT only increased contractile force of colon circular muscle. It was concluded that both aNT and SP may have a role in the regulation of lower gut motility in avian species.     

Absorption of electrolytes and water by the jejunum and colon in milk-fed lambs

Net absorption of electrolytes (Na, Cl, K, Ca) and water from ligated loops was studied at various intestinal sites in milk-fed lambs. The unidirectional fluxes of Na across the intestinal mucosa were also investigated using 22Na. Net Na and water absorption in the mid-jejunum were about two-fold higher than in the proximal and distal jejunum and the colon descendens. With the exception of the proximal jejunum, Na and Cl absorption did not differ significantly. The unidirectional Na fluxes in both directions were much higher in the proximal and mid-jejunum than in the distal jejunum and colon descendens. K was also absorbed most efficiently from the mid-jejunum. In the colon descendens mean net K absorption was about zero. Ca absorption in the upper and mid-jejunum exceeded that of the distal jejunum and colon descendens, where the values were close to zero. The results show that in the whole jejunum of young milk-fed lambs net absorptive fluxes of Na, Cl, K, Ca and water occur, whereas the colon descendens appears to play a role only in Na, Cl and water absorption.     

Oxidative metabolism of rabbit and rat intestine with short chain fatty acids and glucose: an evaluation of data analysis

1. Glucose sustained VO2 of rabbit ileum, caecum, and distal colon better than SCFAs. 2. In rabbit proximal colon, while VO2 was stimulated in the presence of butyrate it was not sustained. 3. Rat caecum utilized glucose but it was not necessarily the best substrate for either the ileum or colon of this species and SCFAs appeared to stimulate VO2 of rat ileum and inhibit VO2 in rat caecum and colon. 4. It was concluded from the comparison of the two methods of data analyses that curve-fitting the data by a negative exponential equation provides for a more clear and in depth interpretation of such studies.     

Contraluminal uptake of serine in the proximal nephron

Rabbit proximal nephron segments were microperfused in vitro to determine whether active contraluminal uptake of serine occurs in the renal proximal tubule during bath-to-lumen transport (influx) of the L- and D-isomers in the convoluted (pars convoluta) and straight (pars recta) segments. It is known that several amino acids are actively reabsorbed in the proximal nephron by a mechanism involving co-transport with sodium at the luminal membrane. There is some evidence that certain amino acids may also be accumulated across the contraluminal membrane by an energy-dependent mechanism, indicating that net reabsorption is the result of two oppositely directed active transport processes. During in vitro microperfusion of rabbit proximal nephron segments in this study, inward movement of L- and D-serine occurred in a bath-to-cell direction against a concentration gradient in the range 305-2735:1, indicating active uptake at the contraluminal membrane. The concentration gradients were maintained during influx of both isomers of serine in the proximal tubule. L-Serine accumulation by tubular cells was similar in the pars convoluta and recta, and significantly greater than that of D-serine, which was the same in both regions of the proximal tubule. The data support the conclusion that renal handling of serine involves active contraluminal uptake of the L- and D-isomers in both regions of the proximal tubule, and suggest that contraluminal events play an important role in renal handling of amino acids.     

Intrarenal localization of degradation of atrial natriuretic peptide in isolated glomeruli and cortical nephron segments

Using isolated glomeruli and nephron segments obtained from collagenase treated rabbit kidneys, we examined the in vitro degradation of alpha-human atrial natriuretic polypeptide (alpha-hANP). The ANP-degrading activity was measured by the amount of immunoreactive ANP remaining after incubation of about 50 fmoles alpha-hANP with each tissue preparation for 7.5 min. The sequence of degrading activity among isolated nephron segments was as follows: proximal straight tubule greater than proximal convoluted tubule greater than cortical collecting tubule greater than distal convoluted tubule greater than cortical thick ascending limb. A single glomerulus exhibited the degrading activity which was comparable to approximately 50% of the activity of 1 mm proximal convoluted tubule. Phosphoramidon, an inhibitor of endopeptidase, prevented the degradation of ANP in proximal convoluted tubule and glomerulus by 68% and 89%, respectively, but not in cortical thick ascending limb and cortical collecting tubule. From these results, we conclude that the degradation of ANP by endopeptidase occurs mainly in the proximal tubule and glomerulus.     

Regional factors affecting proliferation in the large intestine of the rat.

Colonic neoplasia is more frequent in the distal colon than in the proximal colon in spontaneous human disease and in carcinogen-induced tumors in rodents. The possibility that this may reflect regional differences in morphology and in proliferative responses to fasting and refeeding was explored in this study in rats. Scanning electron microscopy revealed that the density of colonic crypts was 36% higher in the distal than in the proximal colon, while light microscopy revealed that distal crypts had 70% more colonocytes than proximal crypts. Thus, the number of colonocytes per unit area in the distal colon is approximately twice that in the proximal colon. Proliferation was assessed by the uptake of bromodeoxyuridine in vivo and showed that regions of the distal colon had greater suppression of proliferation during fasting than the cecum, and greater enhancement of proliferation during refeeding than that observed in the cecum or the proximal colon. Changes in proliferation associated with fasting and refeeding were accompanied by changes in the concentrations of short chain fatty acids, but the data did not support the hypothesis of a direct relationship between increasing concentrations of short chain fatty acids and enhanced proliferation. Regional differences in morphology and proliferation could be relevant to the greater susceptibility of the distal colon to neoplasia.     

Effects of deoxycorticosterone acetate on the electrical properties of rat large intestine: segmental differences

The functional heterogeneity of different segments of the rat large intestine was investigated by means of transepithelial potential difference (PD), short-circuit current (Isc) and transepithelial resistance (Rt) measurements in control rats and after deoxycorticosterone acetate (DOCA) pretreatment. Rt and PD were low in caecum and proximal colon but higher in the distal colon and rectum. Isc was highest in the distal colon, lower in the caecum, proximal colon, and rectum. None of the electrical properties was sensitive to amiloride in control conditions. DOCA increased PD and Isc in the caecum, distal colon and rectum but had no effect in the proximal colon. The increase of the Isc after DOCA in the distal colon and rectum was reached by induction of the amiloride-sensitive Isc associated with reduction of the amiloride-insensitive Isc. The effect of DOCA could be completely prevented by concurrent spironolactone treatment. The results suggest that the epithelia of the proximal parts of the large intestine are "leaky" whereas those of the distal colon and rectum are relatively "tight". It is concluded that there is a marked quantitative and qualitative segmental heterogeneity along the rat large intestine.     

Differential influence of butyrate concentration on proximal and distal colonic mucosa in rats born germ-free and associated with a strain of Clostridium paraputrificum

In vivo influence of butyrate in colonic mucosa was studied using a model of gnotobiotic rats monoassociated with a Clostridium paraputrificum. Rats were fed a diet containing increasing amounts of non-digestible carbohydrates, the fermentation of which led to modulated amounts of butyrate in the large intestine. In the proximal colon, the increase in the butyrate concentration alters crypt depth and the number of mucus-containing cells; the increase in butyrate was highly correlated with the number of neutral-mucin-containing cells. Conversely, in the distal colon, no relation was found between the increase in butyrate concentration and crypt depth or number of mucin-containing cells. In both the proximal and distal colon, the mitotic index remained unchanged. In conclusion, in vivo production of physiological quantities of butyrate had a trophic effect on proximal colonic mucosa, but did not influence the distal epithelium.     

Ion transport by rabbit colon. I. Active and passive components.

Descending rabbit colon, stripped of muscularis externa, absorbs Na and Cl under short-circuit conditions and exhibits a residual ion flux, consistent with HCO3 secretion, whose magnitude is approximately equal to the rate of active Cl absorption. Net K transport was not observed under short-circuit conditions. The results of ion replacement studies and of treatment with ouabain or amiloride suggest that the short-circuit current ISC is determined solely by the rate of active Na transport and that the net movements of Cl and HCO3 are mediated by a Na-independent, electrically-neutral, anion exchange process. Cyclic AMP stimulates an electrogenic Cl secretion, abolishes HCO3 secretion but does not affect the rate of Na absorption under short-circuit conditions. Studies of the effect of transepithelial potential difference on the serosa-to-mucosa fluxes Jism of Na, K and Cl suggest that JNasm,JIsm and one-third of JCl-sm may be attributed to ionic diffusion. The permeabilities of the passive conductance pathway(s) are such that Pk:PNa:PCl= 1.0:0.07:0.11. Electrolyte transport by in vitro rabbit colon closely resembles that reported from in vivo studies of mammalian colon and thus may serve as a useful model for the further study of colonic ion transport mechanisms.     

Unidirectional fluxes of short-chain fatty acids across segments of the large intestine in pig,sheep and pony compared with guinea pig

Unidirectional fluxes of short-chain fatty acids across pig, sheep and pony caecum, proximal and distal colon were studied under short-circuit current conditions in Ussing chambers. Findings are compared with results from guinea pig. Marked species differences are apparent; highest mucosal-to-serosal fluxes of acetate, propionate and butyrate were seen in guinea pig, lower values in pig and smallest fluxes in sheep and pony. Segmental differences between caecum, proximal and distal colon exist mainly in guinea pig and are less developed in pig, sheep and pony. Inhibition of Na⁺/H⁺ exchange by amiloride added to the mucosal solution decreased the mucosal-to-serosal fluxes of short-chain fatty acids clearly in guinea pig caecum and proximal colon, and very little in distal colon. This effect was somewhat less pronounced in pig caecum and distal colon, in caecum and distal colon of sheep and caecum of the pony. In pig, sheep and pony proximal colon and pony distal colon no significant inhibition was observed. Inhibition of the K⁺-H⁺ ATPase by addition of ouabain to the mucosal solution diminished mucosal-to-serosal fluxes of short-chain fatty acids in the guinea pig distal colon extensively. No comparable inhibition was seen in any of the other segments in the animals studied.     

Cationophore properties of the new polyether antibiotic Salinomycin investigated in distal rabbit colon in vivo and in vitro

The distal rabbit colon was used as a model to investigate the influence of the cationophore Salinomycin in vivo with a single-pass perfusion, and in vitro with a modified Ussing chamber technique. For in vivo experiments with labelled 14C-PEG as a volume marker in the perfusate, a dose of 10E-4 mol/l Salinomycin was used. Net water (53 microliters/h/cm), net chloride (3 mumol/h/cm) and net sodium (3.6 mumol/h/cm) absorption was not significantly influenced, but net potassium secretion (-3 mumol/h/cm) was decreased to zero and transepithelial potential (PD) reduced from -45 mV to -33 mV. 10E-4 mol/l Salinomycin, applied in vitro on the muscosal side, decreased PD in 80 min and 10E-3 mol/l in 30 min from 18 mV to zero. Both concentrations decreased the short-circuit current (Isc = 77 microA/cm2) in 60 min, respectively 30 min to 40 microA/cm2. After 60 min mucosal 10E-4 mol/l Salinomycin the Isc increased, resulting from a transepithelial conductance (Gt) increase from 3 to 40 mS/cm2. A dose-related effect was present on PD, Isc and Gt at concentrations between 10E-7 and 10E-6 mol/l. The unidirectional 22Na fluxes were increased to 20 times the control values and net Na transport disappeared. We conclude that Salinomycin when applied in usual doses (10E-4 mol/l) as a coccidiostatic feed additive, profoundly affects colonic electrolyte transport.     

Nippostrongylus brasiliensis: malabsorption in experimentally infected rats

Glucose absorption and net small intestinal water movement were examined in rats infected with Nippostrongylus brasiliensis at Days 4, 6, 9, 13, and 19 after inoculation. Rats were infected with 4 X 10(3) N. brasiliensis third stage larvae. The entire small intestine was divided into three segments and each segment perfused simultaneously in vivo with Krebs-Ringer phosphate buffer containing 80 mM glucose, 6 X 10(5) dpm/ml [3H]glucose, and 6.2 X 10(3) dpm/ml [14C]polyethylene glycol. Rats perfused on Days 6, 9, 13, and 19 after inoculation showed a significant (P less than 0.05) decrease in glucose absorption rates from all three segments of the small intestine when compared to uninfected controls. In the three segments of uninfected rat small intestine and those perfused on Days 4, 13, and 19 after inoculation, net absorption of water occurred. However, in the proximal and distal segments perfused on Day 6 and the proximal segment perfused on Day 9, net water movement into the lumen occurred. This is the first report of depressed glucose absorption along the entire length of the small intestine during nippostrongylosis and contradicts previous reports of unaltered net glucose absorption in response to this parasite.