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Experiments were designed to characterize an active, electrogenic transport of NH(4)(+) ions across the colonic epithelium of the domestic fowl (Gallus gallus). Colonic segments were isolated and stripped of underlying muscle. The mucosal epithelia were mounted in Ussing chambers and voltage-clamped to measure the short-circuit currents (I(SC)) associated with transport. Bilateral addition of NH(4)(+) caused a dose-dependent outward current (negative I(SC)), with a Km of 34+/-8 mM and a maximal current response of 311+/-47 microA cm(-2) (12+/-2 microEq cm(-2) h(-1)). A similar effect was seen with unilateral addition of NH(4)(+) to the serosal (s) side, but not with mucosal (m) addition. Pre-treatment with 10(-4) M amiloride exposed a net outward (negative) I(SC), and serosal NH(4)(+) addition further increased this outward current with a Km of 53+/-24 mM. Decreasing the bath pH from 7.3 to 6.0 did not affect the I(SC) response to NH(4)(+). Unidirectional NH(4)(+) flux measurements revealed a net secretory flux (8.8+/-3.1 micromol cm(-2) h(-1) s-m, versus 2.6+/-1.4 micromol cm(-2) h(-1) m-s). Furthermore, the secretory flux closely matched the resulting change in I(SC) with serosal NH(4)(+), showing that the transepithelial flux of NH(4)(+) could account for the outward current response. Addition of 50 nM bafilomycin A to the mucosal solution completely eliminated serosal to mucosal NH(4)(+) transport, implicating an apical V-type H(+)-ATPase in this transport process. The I(SC) response to NH(4)(+) was partially inhibited by ouabain, a blocker of the Na(+)/K(+)-ATPase, but only minimally affected by bumetanide, an inhibitor of the serosal Na(+)-K(+)-2Cl(-) cotransporter. Active NH(4)(+) extrusion across the mucosal membrane, combined with low permeability to NH(3) in this tissue, allow for maintenance of steep ammonia gradients across the colonic epithelium and protection from ammonia toxicity. Furthermore, these studies indicate that the hen colon may be a useful new model system for the study of NH(4)(+) transport.

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.

Two antisecretory factor (AF)-derived peptides have been studied in relation to effects on motility of guinea pig colon. Colon segments were isolated from adult guinea pigs and incubated in Tyrode Ringer. Motility was measured as the force and frequency of contractions upon addition of the derived peptides AF 1 (8 amino acids (aa)) and AF 3 (10 amino acids). At the lowest concentration (5 pM), peptide AF 1 induced a negative effect on the force of contraction in colon segments; an effect that was abolished by the cholinergic agonist carbachol. Peptide AF 3 induced a significant increase in the force of colon contractions at all concentrations (5-180 pM), with carbachol only reducing the effect of peptide AF 3 at a concentration of 15 pM. Both peptides increased contractile frequency, although the overall response was lower for peptide AF 3 than for peptide AF 1. It is concluded that antisecretory factor-derived peptides may play a role in regulating colon motility such that under pathophysiological conditions, they may serve to hasten the evacuation of noxious agents from the large intestine.

1. Occurrence of short chain fatty acids (SCFA) in anionic form limits their diffusion across the absorptive membrane. The present study sought to establish the mechanism of SCFA absorption in the ostrich. 2. Epithelial tissues were taken from the sacculated part of the colon and mounted in Ussing chambers in a bathing solution. The tissues were voltage-clamped and allowed to equilibrate to obtain a baseline short circuit current (Isc). 3. Propionate (23 mM) on the mucosal side increased the Isc. The SCFA-induced Isc was completely inhibited by anoxia, ouabain (1 to 2 mM), acetazolamide (0.5 mM) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (0.1 mM) on the mucosal side. 4. These findings indicate that SCFA stimulate hydrogen ion secretion through an electrogenic H(+)-K(+)-ATPase, the source of hydrogen ions being carbonic anhydrase catalysed hydration of CO2. 5. Simultaneous activation of Cl(-)/HCO3(-) exchange prevents intracellular accumulation of bicarbonate ions. This system may provide hydrogen ions for protonation of SCFA anions and subsequent absorption by non-ionic diffusion.

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.

Experiments were performed on isolated, stripped colonic epithelia of low-salt-adapted hens (Gallus domesticus) in order to characterize acid secretion by this tissue. With symmetric, weak buffer solutions, colonic epithelia acidified both mucosal and serosal sides. Titration measurements of the mucosal acidification rate (pH-stat technique) averaged 1.63 +/- 0.25 microEq.cm-2.h-1. Mucosal acidification was also evident in colons from high-salt-adapted birds and in low-salt-adapted coprodeum, but was completely abolished in the high-salt coprodeum. Mucosal acidification by low-salt-adapted colonic epithelium was unaffected by sodium replacement, mucosal amiloride (10(-3) mol.l-1), and serosal ouabain (5 x 10(-4) mol.l-1), although all three treatments significantly reduced or reversed the short-circuit current. Acetazolamide (10(-3) mol.l-1, serosal) reduced mucosal acidification by 15% and simultaneously increased short-circuit current by a similar amount. Colonic epithelia incubated in glucose-free solutions had significantly lower acidification rates (0.59 +/- 0.13 microEq.cm-2.h-1, P < 0.002 versus controls) and addition of glucose (15 mmol.l-1), but not galactose, partially restored acidification to control levels. Anoxia (N2 gassing) completely inhibited short-circuit current, but reduced acidification by only 30%. A surface microclimate pH, nearly 2 pH units more acidic than the bath pH of 7.1-7.4, was measured in low-salt-adapted colon and coprodeum. The acid microclimate of both tissues was partially attenuated by adaptation to a high-salt diet. Colonic microclimate pH was dependent on the presence of glucose and sensitive to the bath pH.(ABSTRACT TRUNCATED AT 250 WORDS)

We have used polyclonal antibodies generated against purified bovine renal amiloride-sensitive Na+ channels to localize amiloride-sensitive Na+ channels within the lower intestine (colon and coprodeum) of the hen. These antibodies cross-reacted with two polypeptides exhibiting M(r)'s of 235 and 150 kDa on immunoblots of detergent-solubilized apical membrane fractions from both the colon and coprodeum. The apparent molecular masses of theses polypeptides are in agreement with the M(r)'s of 2 of the subunits of the renal high amiloride-affinity Na+ channel, namely the alpha and the beta (= amiloride binding) subunits. The cellular distribution of Na+ channels was determined by immunoperoxidase and indirect immunofluorescence cytochemical techniques. The apical (luminal) membrane and cytoplasm of villar principal cells in both colon and coprodeum exhibited immunoreactivity, whereas goblet cells were negative. Both principal and goblet cells of the crypts were also negative. We conclude that the amiloride-sensitive Na+ channels are localized to the principal cells of the intestinal villi and that these cells are responsible for intestinal Na+ absorption.

1. Short-chain fatty acid absorption in hen colon is protonated across the apical border coupled to an apical electrogenic proton pump. 2. The surface pH of the isolated colonic epithelium was 6.27 +/- 0.05, when incubated in Krebs-phosphate buffer pH 7.0. 3. Propionate 7 and 40 mmol/l in the incubation medium (pH 7.0) increased microclimate pH to 6.47 +/- 0.04 and 6.56 +/- 0.04. Inhibition of metabolic activity by potassium cyanide 1 mmol/l increased surface pH to 6.66 +/- 0.06. 4. The calculated concentration of propionic acid in the microclimate is near-linearly related to the propionate concentration. Thus, the acid microclimate is not responsible for the Michaelis-Menten like kinetics of propionate transport.

The regulation of Na and Cl transport in hen colon by mineralocorticoids was investigated with isolated epithelia in vitro by histological and electrophysiological techniques. The electrogenic transport of Na and Cl was measured in Ussing chambers by the short-circuit current technique and was identified by the specific inhibitors amiloride and bumetanide or by the secretagogue theophylline. Hens were maintained either on low (LS)- or on high-NaCl diets (HS), and the plasma aldosterone (PA) levels of these groups were measured with radioimmunoassay. A group of HS hens received injections of aldosterone at a 6-h schedule before experiments. A group of LS hens was resalinated, and experiments were carried out at a 24-h interval for up to 3 days after resalination. The LS diet stimulated PA levels ninefold, compared with HS hens. Na transport was modulated by the hormonal stimulus in a way that the apical Na entry switched from an electrogenic Na-amino acid-hexose cotransport system completely to an amiloride-sensitive Na channel. Electrogenic Cl secretion was induced by theophylline and was inhibited by bumetanide. NaCl deprivation, resalination or aldosterone injection modulated electrogenic Cl secretion in parallel between 7 (HS) and 14.4 mu eq.cm-2.h-1 (LS), with pronounced alteration in tissue conductance. These findings reveal a new action of aldosterone that, besides stimulating Na absorption, also directly or indirectly modulates Cl secretion.

The colon and coprodeum of anaesthetised laying hens was perfused luminally with solutions of varying compositions, to elucidate the influence of three levels of dietary NaCl and of luminal NaCl concentrations on coprodeal and colonic transport of NaCl. Net Na+ and Cl- absorption rates were increased in response to low dietary Na+ levels and higher luminal NaCl concentrations. Net Na+ absorption was always against the prevailing electrochemical gradient, saturably dependent on luminal Na+ concentrations in low Na+ diet birds, and linearly dependent on luminal concentrations in medium and high Na+ diet birds. Net Cl- absorption was usually down its electrochemical gradient. The Cl- flux rates were strongly linearly dependent on Na+ flux rates, and statistically this relationship could account for all significant variation of Cl- fluxes associated with dietary and perfusate treatments. Transmural electrical potential differences were invariably serosa positive, and partly correlated with Na+ fluxes.

The lower intestine (coprodeum and colon) of the Australian parrot, the galah, was mounted in Ussing chambers. Short-circuit current (SCC), electrical potential difference (PD), and unidirectional fluxes of Na and Cl were measured in birds that were fed mixed seeds or were NaCl loaded. The net Na transport of both coprodeum and colon was nearly equal to the SCC, and the flux ratio for Cl was unity. In birds which received mixed seeds, average coprodeal Na transport was 7.8 mu eq . cm-2 . h-1, and PD was 19 mV. The Km for Na was 5.7 meq/l. In colon, Na transport was reduced by 67% and PD by 70%. The ratio between unidirectional Na and Cl fluxes in the serosa-mucosa direction was 0.7. Salt loading suppressed coprodeal, but increased colonic Na transport. The coprodeal and colonic SCC and NA transport of birds receiving mixed seeds were inhibited by amiloride on the mucosal side. Colonic SCC of NaCl-loaded birds was only slightly reduced by amiloride (by 17%), but stimulated by amino acids (by 18%).

1. Sheets of isolated mucosa from the colon of chickens on either a high or a low NaCl diet were mounted in Ussing chambers. They were bathed in Krebs-phosphate medium and exposed to sodium concentrations ranging from 3.0 to 140 mmol/l (choline-chloride replacement). 2. In the short-circuited state the net fluxes of sodium in the mucosa (m)-serosa (s) direction, followed saturation kinetics with Km = 25 mmol/l in the low-NaCl chickens and 124 mmol/l in the high-NaCl chickens. 3. The unidirectional fluxes of sodium in the s-m direction were linearly related to the electro-chemical driving force. The apparent permeabilities were 8.6 +/- 0.3 x 10(-6) cm/s in the low-NaCl and 11.6 +/- 0.3 x 10(-6) cm/s in the high-NaCl birds. 4. The chloride fluxes were in the short-circuited state of equal magnitude in the m-s and the s-m direction, and in the two dietary states. Changes in the electrical driving force resulted in changes in chloride fluxes compatible with passive flow.

The effects of pre-treatment in vivo with aldosterone and dexamethasone were investigated on rabbit distal colon. Apical Na-permeability and net sodium transport were measured in vitro. In this epithelium, Na-transport is entirely electrogenic. It can therefore be measured electrically as the fraction of short circuit current which is blockable by amiloride. The epithelia were studied in an Ussing chamber and the electrical values recorded by a computerized digital voltage clamp. Transepithelial parameters, and the transapical membrane parameters (in preparations depolarized from the serosal side) were investigated after treatment with the two hormones. Under transepithelial conditions, aldosterone and dexamethasone stimulated the short circuit current (Isc) from control (17.4 microA/cm2) to a similar degree (86.6 and 93.8 microA/cm2). However, whereas aldosterone did not alter the transepithelial resistance (RT) significantly, dexamethasone reduced RT from 357 to 167 omega X cm2. The stimulation of the potential difference (VT) under control condition (6.6 mV) was therefore significantly different between aldosterone (28.7 mV) and dexamethasone (16 mV). Mucosal amiloride (0.1 mM) inhibited Isc and VT completely under all conditions. Steady state current-voltage relations were obtained by voltage clamping the tissues in "staircase" increments before and after mucosal treatment with amiloride. As measured by the difference between these two states, Na-currents were calculated both for the transepithelial and the transapical condition. Intracellular Na-activity and apical Na-permeability were then calculated by the Nernst and Goldman-Hodgkin-Katz equations. These values were found to be increased after treatment with both hormones. Dexamethasone was a more potent stimulator of both values.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Variations in renal microstructure between the zebra finch and Senegal dove were consistent with their relative renal concentrating abilities (urine/plasma ratios of 2-8 and 1-7, respectively). Compared with dove kidneys, those of the finch contained a higher fraction of mammalian-type nephrons (with Henle's loops), and a lower fraction of reptilian-type nephrons (without loops). 2. Singing honeyeaters concentrated their urine almost as well as zebra finches, although honeyeater kidneys were less specialized (fewer mammalian-type nephrons). Such findings emphasize the need to clarify other osmoregulatory parameters. 3. No significant microstructural differences were found in the kidneys of domesticated as compared with those of wild zebra finches. Hence, osmoregulatory differences between tame and wild birds must be related to physiological factors rather than morphological. 4. Thickness of the renal medulla seemed to be directly correlated with urine concentrating ability. However, certain inconsistencies obscure this relationship such that its resolution will require further research. 5. Histological features of the mucosae of the colon and cloaca are described. The galah and kookaburra displayed a mammalian (non-villous) pattern of mucosal organization. Zebra finches, singing honeyeaters, and particularly emus, possessed colonic and cloacal villi and hence an increased surface area per volume in this region of the gut. This raises the possibility that the colon and cloaca are involved in uring concentration and osmoregulatory activities in these species.

1. The isolated colonic mucosa from adult hens has been studied in vitro under short-circuit conditions. Colonic mucosa was prepared from hens fed either a NaCl-poor or a NaCl-rich diet. 2. The unidirectional transmucosal fluxes of chloride, K and Na were measured. For the high NaCl colon the fluxes of lysine were also measured. The effects of galactose, glucose, leucine, lysine, amiloride (10(-4) M) and acetazolamide (5 X 10(-4) M) on the short-circuit current (Isc) were examined. 3. The Isc of the high NaCl colon was stimulated by galactose, glucose, leucine and lysine in a manner which suggests that these substances were transported by the epithelium in co-transport with Na. The Isc was in most cases insensitive to amiloride (mucosal side), but moderately stimulated by acetazolamide (serosal side). The Isc was closely matched by the sum of the net transmucosal fluxes of chloride, lysine, K and Na. The effects of galactose, leucine and lysine on the Isc were maintained only when glucose was present on the serosal side of the preparations. 4. The Isc of the low NaCl colon depended on the presence of glucose on the serosal (or mucosal) side. It was not stimulated by galactose or by lysine, but leucine did stimulate when added to the mucosal solution. Adding amiloride on the mucosal side invariably eliminated or, in most cases, led to an inversion of the Isc. Acetazolamide markedly stimulated the Isc; and, when added after amiloride, acetazolamide caused the Isc to become positive again. The data on Isc and net fluxes of chloride, lysine, K and Na are consistent with the presence of a process of bicarbonate absorption or H ion secretion. 5. There is a net secretion of K across both the high and the low Na colon, which is highest in the former although both Isc and net absorption of Na are highest in the latter. 6. The results from both types of colon suggest that the transport of chloride differs with the functional status of tissues.

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.

The binding of 3H-benzamil to homogenates of epithelium removed from the colon and coprodaeum of hens was studied. A low capacity high affinity binding component was detected. The binding constants for benzamil and amiloride to this component were similar to those obtained when these ligands were used to inhibit transport in intact epithelia. The mean potency ratio of benzamil to amiloride was 12.8 measured by binding compared with 11.6 from functional inhibition. Binding activity was present in tissues taken from animals fed on low sodium diets and those containing a normal sodium content. In the presence of sodium the affinity of benzamil was slightly reduced, but only in tissues taken form animals on a low salt diet. Only a small fraction of the total binding activity was present in the apical surface of low salt tissues indicating that in homogenates a small percentage of the total activity is associated with functional sodium entry sites. It is suggested that the major part of the binding activity detected in homogenates represents components of the sodium ion translocation mechanism which are en route for the apical membrane.

The colon and coprodeum of anaesthetised laying hens was perfused luminally with solutions of varying compositions to elucidate the influence of three levels of dietary Na+ and of luminal fluid composition on transport of electrolytes and water. Net rates of secretion of K+ and absorption of NH4+ were increased in response to dietary Na+ depletion and increased luminal Na+ concentrations, but these fluxes were linked to Na+ transport and no dietary or perfusion treatment effects could be detected on them independently of effects on Na+ flux. Similar results were found for Na+-linked water absorption from isosmotic perfusates. Serosal-mucosal net osmotic flow with Na+-free perfusates was significantly reduced by Na+-depletion, but no significant dietary effect could be detected on the serosal-mucosal osmotic permeability coefficient. The reflexion coefficient was effectively unity. Mucosal-serosal inorganic phosphate flux was small. The flux was independent of Na+ flux and dietary Na+ levels, and apparently facilitated by serosalmucosal osmotic flow. The data allowed determination of the absorption/secretion in coprodeum and colon of the ions investigated, as compared to the renal excretion rate.

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.