Serosal Medium Research Articles

Effect of calcium on amino acid and dipeptide transport by intestines of American lobster (Homarus americanus) and Atlantic white shrimp (Litopenaeus setiferus) (908.6)

Previous work using L‐histidine and zinc in American lobster intestine has shown these solutes can be transported as bis‐complexes (Histidine‐Zinc‐Histidine) across the intestine using a peptide transporter. Furthermore, transport of L‐leucine has been shown to be inhibited by high calcium concentrations. Dipeptide and bis‐complex transport and the role of calcium were investigated in the perfused intestine of lobster and Atlantic white shrimp. Samples of serosal medium were analyzed for amino acid composition by gas chromatography. In lobster, the transport of glycylsarcosine (Gly‐Sar) from mucosal to serosal medium was measured for a range of pH values and either 0.5mM or 25mM calcium chloride. The rate of Gly‐Sar transport was stimulated two‐fold with pH 8.5 in the lumen. Reduced calcium chloride concentration stimulated Gly‐Sar transport two‐fold. In shrimp, the transport of L‐histidine from mucosal to serosal medium was measured. The addition of 50µM zinc chloride increased the rate of histidine transport, while Gly‐Sar inhibited transport. The rate of histidine transport was significantly higher with 1mM calcium chloride than with 25mM. These results indicate that shrimp transport bis‐complexes in a manner similar to lobster, which is inhibited by the presence of a competing dipeptide. High calcium concentration has an inhibitory effect on both amino acid and dipeptide transport.Grant Funding Source: Supported by USDA Agricultural and Food Research Initiative Competitive Grant no. 2010‐65206‐20617

Airway Epithelial Inflammation-induced Endoplasmic Reticulum Ca2+ Store Expansion Is Mediated by X-box Binding Protein-1

Inflamed cystic fibrosis (CF) human bronchial epithelia (HBE), or normal HBE exposed to supernatant from mucopurulent material (SMM) from CF airways, exhibit endoplasmic reticulum (ER)/Ca(2+) store expansion and amplified Ca(2+)-mediated inflammation. HBE inflammation triggers an unfolded protein response (UPR) coupled to mRNA splicing of X-box binding protein-1 (XBP-1). Because spliced XBP-1 (XBP-1s) promotes ER expansion in other cellular models, we hypothesized that XBP-1s is responsible for the ER/Ca(2+) store expansion in inflamed HBE. XBP-1s was increased in freshly isolated infected/inflamed CF in comparison with normal HBE. The link between airway epithelial inflammation, XBP-1s, and ER/Ca(2+) store expansion was then addressed in murine airways challenged with phosphate-buffered saline or Pseudomonas aeruginosa. P. aeruginosa-challenged mice exhibited airway epithelial ER/Ca(2+) store expansion, which correlated with airway inflammation. P. aeruginosa-induced airway inflammation triggered XBP-1s in ER stress-activated indicator (ERAI) mice. To evaluate the functional role of XBP-1s in airway inflammation linked to ER/Ca(2+) store expansion, control, XBP-1s, or dominant negative XBP-1 (DN-XBP-1) stably expressing 16HBE14o(-) cell lines were used. Studies with cells transfected with an unfolded protein response element (UPRE) luciferase reporter plasmid confirmed that the UPRE was activated or inhibited by expression of XBP-1s or DN-XBP-1, respectively. Expression of XBP-1s induced ER/Ca(2+) store expansion and potentiated bradykinin-increased interleukin (IL)-8 secretion, whereas expression of DN-XBP-1 inhibited bradykinin-dependent IL-8 secretion. In addition, expression of DN-XBP-1 blunted SMM-induced ER/Ca(2+) store expansion and SMM-induced IL-8 secretion. These findings suggest that, in inflamed HBE, XBP-1s is responsible for the ER/Ca(2+) store expansion that confers amplification of Ca(2+)-dependent inflammatory responses.

Bioelimination of histamine in epithelia of the porcine proximal colon of pigs

The study aimed to gain insight into how intestinal histamine N-methyltransferase (HMT) and diamine oxidase (DAO) could contribute to bioelimination of histamine. Mucosal-to-serosal (ms) and serosal-to-mucosal (sm) fluxes of histamine, choline or 5-hydroxytryptamine were measured in isolated colonic epithelia of pigs (Ussing chambers). Radioactively (hist-rad) and HPLC-measured histamine fluxes were higher in sm vs. ms direction at 100 microM histamine. Choline (3-3000 microM) and 5-HT (20 microM) fluxes only tended to be higher in sm vs. ms direction. Secretion of hist-rad was abolished by serosal 1-methyl-4-phenylpyridinium (MPP). Histamine fluxes accounted for <25 % of hist-rad fluxes, but this percentage increased after blocking HMT (100 microM amodiaquin) or DAO (100 microM aminoguanidine). 1-Methylhistamine (1-MH) appeared exclusively in the serosal medium. 1-MH appearance decreased after addition of amodiaquin or after addition of N-ethylmaleimide (1 mM NEM). Blockage of vesicular trafficking by NEM enhanced histamine catabolism, which could be reversed by aminoguanidine. DAO was detected in punctuate structures in the basal parts of colonocytes by immunohistochemistry. A basolateral organic cation transporter facilitates histamine secretion into the intestinal lumen and delivers histamine to catabolism by HMT and/or vesicular DAO. Histamine is partially released back into the blood after initial biotransformation to 1-MH.

Regulation of urea permeability in frog urinary bladder by prostaglandin E(2).

The present study was performed to investigate the role of prostaglandin E(2) (PGE(2)) in the regulation of urea transport in the frog urinary bladder, which is known to occur via a specialized arginine-vasotocin- (AVT-) regulated urea transporter. The bladders isolated from Rana temporaria L. were filled with amphibian Ringer solution containing 370 Bq/ml (0.01 microCi/ml) of [14C]urea, and urea permeability ( P(urea)) was determined by sampling the serosal and mucosal bathing medium at 30-min intervals for measurement of radioactivity. It was found that, from the serosal side, PGE(2) (10 nM to 1 microM) caused a dose-dependent increase in P(urea) [(7.2+/-1.8)x10(-6) cm/s in the presence of 0.5 microM PGE(2)versus (1.0+/-0.2)x10(-6) cm/s in control, n=9, P<0.001]. As in response to AVT, the PGE(2)-induced P(urea)reached a maximum in 1-1.5 h after the agonist was added. The stimulatory effects of PGE(2) and AVT applied together were not additive. PGE(2)-induced urea transport was strongly inhibited by nearly 75% in the presence of mucosal or serosal phloretin (10(-4) M). P(urea) was enhanced up to (4.7+/-0.8)x10(-6) cm/s (n=12, P<0.001) by butaprost (5 x 10(-6) M), a selective EP(2) receptor agonist, while sulprostone (EP(1)/EP(3) agonist, 10(-6) M) caused no changes in P(urea). PGE(2)dose-dependently increased the content of cAMP in mucosal epithelial cells (control: 18.0+/-1.8; 10(-6) M PGE(2): 74.2+/-9.3 pmol cAMP/mg protein per 30 min, n=7, P<0.001). Phorbol esters did not alter PGE(2)-induced P(urea), whereas H-89 (20 microM), a protein kinase A inhibitor, reduced it by 45.1+/-9.9% ( n=5, P<0.05). PGE(2)did not change the AVT-stimulated P(urea) measured in isoosmotic conditions, but inhibited the last one in the presence of a serosa-to-mucosa osmotic gradient. The data obtained show that, in the frog urinary bladder, PGE(2)is a stimulator of phloretin-inhibitable urea transport. Its effect seems to be mediated by EP(2) receptor-coupled generation of intracellular cAMP.

Various Indigestible Saccharides Enhance Net Calcium Transport from the Epithelium of the Small and Large Intestine of Rats In Vitro

An Ussing chamber technique was used to determine the effects of six indigestible saccharides on net Ca absorption from the luminal side to the basolateral side of isolated preparations of rat jejunal, ileal, cecal and colonic epithelium in vitro. The concentrations of Ca in the Tris buffer solution on the serosal side and on the mucosal side were 1.25 and 10 mmol/L, respectively. After a 30-min incubation, the Ca concentration in the serosal medium was determined and the net transepithelial Ca transport was calculated. The addition of 0.1-200 mmol/L maltitol, difructose anhydride (DFA)III, DFAIV, raffinose, fructooligosaccharide (FOS) or polydextrose (PD) to the mucosal medium increased the net Ca absorption dose-dependently in the jejunum, ileum, cecum and colon preparations. The threshold concentration required to enhance Ca transport and the extent of enhancement of Ca transport differed among the saccharides tested and among the portions of the intestine examined. Among the saccharides tested, DFA IV had the strongest effect on Ca absorption in the jejunum and cecum. We conclude that indigestible carbohydrates directly affect the epithelial tissue and promote Ca absorption in both the small and large intestine in vitro.

Short-chain fatty acids enhance diffusional Ca transport in the epithelium of the rat cecum and colon

We examined the effect of short-chain fatty acids (SCFAs) on Ca absorption from the large intestine in rats in vitro. An Ussing-type chamber technique was used to determine the net transport of Ca from the luminal side to the basolateral side of isolated epithelium in cecum and colon preparations. The concentration of Ca in the serosal and mucosal Tris buffer solution was 1.25 mM and 10 mM, respectively. Both solutions were warmed at 37 °C and bubbled with 95%O 2 and 5%CO 2. During and after the incubation period (30 min or 60 min), the Ca concentration in the serosal medium was determined and the net transepithelial Ca transport was evaluated. The addition of 80 mM acetic acid, 40 mM propionic acid and 10 mM butyric acid to the mucosal medium increased net Ca absorption (about 300%) in the cecum and colon. An individual application of acetic, propionic or butyric acid (0.01 to 100 mM) to the mucosal medium also increased net Ca absorption at doses of 10 mM and /or 100 mM in the cecum and colon. An increase in solute concentration in the mucosal medium by addition of glycerol or PGE400, or a decrease in pH (7.0–3.0) by addition of HCl did not affect transepithelial Ca transport. We concluded that SCFAs affect the epithelial tissue and promote Ca absorption from the large intestine in vitro. The enhancement of Ca transport induced by SCFAs might be involved in the paracellular transport mechanism.

Ingestion of the soluble dietary fibre, polydextrose, increases calcium absorption and bone mineralization in normal and total-gastrectomized rats

We previously demonstrated that feeding a highly fermentable and water-soluble dietary fibre, guar-gum hydrolysate (GGH) increased intestinal absorption of insoluble Ca salts in total-gastrectomized rats. In the present study, we examined the effects of feeding a less fermentable and water-soluble fibre, polydextrose (PD), on Ca absorption and bone mineralization in the normal and total-gastrectomized rats in comparison with the effects of GGH. Apparent Ca absorption was severely lowered by gastrectomy, and PD feeding (50 g/kg diet) partially restored the reduction of Ca absorption similarly to GGH feeding (50 g/kg diet). PD feeding also increased the Ca absorption in normal rats, but not GGH feeding. Femur Ca concentration was reduced with gastrectomy. Feeding PD for 21 d increased the bone Ca concentration in both normal and gastrectomized rats, but GGH feeding did not. In rats fed PD, pH of the caecal contents was lower than in rats fed fibre-free and GGH diets; however, soluble Ca concentration in the caecal contents was not different between the diet groups. Short-chain fatty acid concentrations were much lower in the PD groups than in the GGH groups. We also examined in vitro Ca absorption by using everted sacs of the small intestine. Addition of PD to the serosal medium of the ileal sacs increased Ca absorption, but addition of GGH did not. These results suggest that the small intestine rather than the large intestine is responsible for the increase in Ca absorption in rats fed PD, and suggests that the mechanism for the increase by PD may be different from that by GGH.

Alpha1-adrenoceptors antagonize activated chloride conductance of amphibian skin epithelium.

The effects of adrenoceptor agonists on the transepithelial Cl- conductance (GCl) in the skin of several amphibian species, both toads and frogs, were studied. Epinephrine (Epi) from the serosal side selectively and reversibly inhibited the voltage-activated GCl in toad skin and the short-circuit GCl in frog skin. The main effects of activation of the adrenoceptors must reside in the skin epithelium and not in the glands, since measurements were made both from intact skins and split epithelia with essentially the same results. Effective concentrations of Epi were variable among individual tissues. GCl was reduced to 34+/-17% (n=46) with 1 micromol/l Epi, but in some tissues 0.1 micromol/l inhibited more than 80% of GCl, whereas some preparations were little influenced at >3 micromol/l Epi. The affected receptor type was identified by the use of the alpha1-agonist phenylephrine, which mimicked the response of Epi at concentrations above 30 micromol/l, whereas the alpha2-agonists xylazine and iodoclonidine had no effect at supramaximal concentrations. Prazosin, a specific alpha1-antagonist, reduced or eliminated the inhibition by Epi, but the response pattern suggests a low affinity. The alpha2-antagonist yohimbine, at concentrations </=0.3 micromol/l, had a minimal effect, but reduced the inhibition by Epi at concentrations of 1-10 micromol/l. This might indicate affinity to alpha1-adrenoceptors in amphibian skin. Activation of beta-adrenoceptors by isoproterenol (0.1-5 micromol/l) led to a transient increase of the baseline inactivated component of GCl with a slight reduction of the voltage-activated GCl at the higher concentrations, but the inhibitory effect of Epi was not altered. Epi, on the other hand, neither prevented nor reversed the induction of a voltage-insensitive GCl in toad skin caused by application of cAMP at supramaximal concentrations (>100 micromol/l CPT-cAMP). Preincubation of the serosal medium with Ca2+-free solution (in the presence of 2 mmol/l EGTA) for extended periods of time (>30 min) eliminated the response to Epi. It is concluded that alpha1-adrenoceptors participate in the physiological control of voltage-activated Cl- conductance in amphibian skin epithelium via modulation of intracellular Ca2+, presumably by efflux from intracellular stores.

Role of prostaglandin E 2 in regulation of low and high water osmotic permeability in frog urinary bladder

The water osmotic permeability of frog urinary bladder was found to be increased from 0.08±0.01 to 1.28±0.20 μl/min cm 2 when serosal bathing medium was changed 4 times for a fresh Ringer solution. High epithelium permeability is accompanied by an increased content of cyclic AMP in the bladder tissue (by 42%, P<0.01), higher activity of both basal and forskolin-stimulated membrane adenylate cyclase (AC) (by 109% and 74%, respectively, P<0.05) and by appearance of aggregates of intramembranous particles in the apical membrane. The water flow was inhibited by 10 −9–10 −5 M prostaglandin E 2 (PGE 2); the inhibitory effect was eliminated in the presence of 10 −4 M N-ethylmaleimide. The increase of water permeability due to changes of the bathing medium was accompanied by a decrease of serosal PGE 2 concentration from 14.8±1.0 in the 1st solution to 0.6±0.1 nM in the 5th. 10 −6 M PGE 2 in vitro inhibited the activity of membrane AC from highly permeable bladders by 33.4% ( P<0.02). Pretreatment of the membranes with 10 μg/ml pertussis toxin (PT) completely reversed this effect (+149%, P<0.01). A significant activation of AC was also observed under 10 −10 M PGE 2 (by 196%). These data demonstrate that the water permeability could be markedly increased independently of ADH, suggesting that the trigger role in activation of water transport is played by a decreased level of PGE 2 which could stimulate AC.

Ammonia transport by the urinary bladder of Bufo marinus.

All experiments were performed in vitro on toad bladders. Bladder sacs from acidotic toads produced a concentration gradient across the bladder with both [NH3] and [NH4] higher in the mucosal media. By varying the pH of the serosal media, paired sacs from normal toads were incubated with similar [NH3] in the serosal media but a 75 fold difference in [NH4] of the serosal media of the pairs. The hemibladders with the higher [NH4] had a 2.4 fold greater excretory flux than the paired sac. Both serosal to mucosal and mucosal to serosal fluxes were determined in normal bladders between chambers at various ammonium concentration gradients. The plot of mucosal to serosal flux against concentration produced a curve compatible with both carrier mediated and diffusion transport; the plot of serosal to mucosal flux produced a straight line with flux increasing when the ammonium concentration was increased. The serosal to mucosal transepithelial flux was augmented by making the serosal side of the bladder 50 mV positive. Although NH3 diffusion may occur, it cannot be the primary method of ammonia transport in the toad bladder.

Effects of barium Chloride on electrolyte transport across isolated colons from normal and Aldosterone-treated lizards (Gallotia Galloti)

Addition of BaCl2 to the solution bathing colons of normal lizards did not alter the absorptive Na+ flux, but did reverse the net absorption of Cl- to become net secretion. Cl- secretion resulted from an increase in its serosal-to-mucosal movement and was positively correlated to an increase in short-circuit current. Ba2+-induced short-circuit current was dependent on the presence of both Na+ and Cl- in the serosal medium. Ba2+-induced Cl- secretion could be reversed by serosal amiloride (10(&shy;4) mmol l-1). Colons from acutely or chronically aldosterone-treated lizards exhibited a considerably higher short-circuit current, potential difference and net Na+ absorption than did untreated colons. Net Cl- transport was unaltered by acute treatment, but was totally abolished after chronic treatment. BaCl2 rapidly decreased the potential difference, short-circuit current and tissue conductance across colons from aldosterone-treated lizards. Net Na+ transport was markedly inhibited by Ba2+ in both acutely and chronically treated tissues, but barium did not change unidirectional or net Cl- fluxes in these. The present results support the following hypotheses: (1) that BaCl2 inhibits electrogenic Na+ absorption induced by acute or chronic aldosterone treatment and (2) that Ba2+ induces an electrogenic Cl- secretion by stimulating a basolateral Na+-dependent Cl- intake in normal but not in aldosterone-treated colons. This also suggests that aldosterone could exert an antisecretory influence in colonic epithelia.

Effect of Phaseolus vulgaris lectins on glucose absorption, transport, and metabolism in rat everted intestinal sacs

Many legume seeds contain lectins. Some of these lectins are toxic when fed to humans or laboratory animals. Although the mechanism for such toxicity has not been clearly elucidated, lectin binding to the intestinal mucosa is an obligatory step. Therefore, the interaction of lectin and enterocytes may interfere with the digestive process. Lectins from toxic (red kidney bean) and non-toxic (mountaineer half runner) Phaseolus vulgaris varieties were tested for their effect on the intestinal absorption, transport, and metabolism of glucose. Both lectins were purified by affinity chromatography on Con A-Sepharose 4B. Everted sacs, 5 cm long, from the first third of the small intestine were incubated for 20 min in the presence of 10 mmol/L glucose in both the luminal and serosal media. Pre-incubation (15 min) of the everted sacs with increasing concentrations of the red kidney bean lectin (50–200 μg/mL mucosal solution) before glucose addition reduced both the absorption and transport of the sugar from the luminal to the serosal side. Glucose metabolism as judged by lactate formation was not affected. At a concentration of 100 μg/mL of red kidney bean lectin, absorption, and transport were inhibited by 40% and 72%, respectively. Lactate production was decreased by only 5%. Under the same conditions (100 μg/mL) mountaineer half runner lectin inhibited glucose absorption and transport by less than 10%. Interference of lectins with the absorption and transport of nutrients could partially explain the toxic effect of lectins from some Phaseolus vulgaris varieties.

Sodium-dependent short-circuit current across the yolk sac membrane during embryonic development in normal and shell-less cultured chicks.

The transepithelial electrical characteristics of the isolated yolk sac membrane of normal in ovo or shell-less cultured chick embryos were investigated. In normal chicks the potential difference (blood side positive relative to yolk side) and short-circuit current of the membrane increased during development. Ouabain (10(-4) M) on the blood side (basolateral side, serosal side) significantly decreased potential difference and short-circuit current but was without effect on the yolk side (brush border side, mucosal side). Substitution of choline for Na+ in the bathing solutions abolished the potential difference and the short-circuit current; when Na+ replaced choline this effect was reversed. Amiloride added to both sides of the yolk sac membrane had no effect on potential difference or short-circuit current. Injection of aldosterone (50 micrograms) and T3 (10 microM) into yolk did not induce amiloride sensitivity. The short-circuit current was not altered by addition of either glucose or alanine to the bath. The short-circuit current of the yolk sac membrane of shell-less cultured embryos was significantly lower than that of normal controls. Addition of Ca2+ to the serosal bathing medium did not reverse the foregoing condition, but decreased the short-circuit current. It is concluded that the yolk sac short-circuit current is Na+ dependent and increases with developmental age in the chick embryo.

A possible relationship between KCl symport and basolateral K +-conductance in Necturus gallbladder epithelial cells

1. 1. Apical membrane potential ( V a), transepithelial potential ( V T), fractional apical voltage ratio ( FV a = ΔV a/ ΔV T), tissue resistance ( R T), and intracellular Cl − ( a Cl i) and K + ( a K i) activities were measured in isolated gallbladders maintained between oxygenated bicarbonate-free physiological media (23°C, pH 7.2 or 8.2) in a divided chamber. The basolateral membrane potential ( V b) was calculated from the measured values of V a and V T. 2. 2. Cl − removal from the serosal medium (which should accelerate coupled basolateral KCl exit) significantly depolarized V b, decreased a Cl i, decreased FV a, increased R T, and attenuated the depolarization of V b ( ΔV b) induced by high K + added to the serosal side. These changes are consistent with a decrease in the K +-conductance of the basolateral membrane ( g K b). 3. 3. Addition of furosemide (an inhibitor of KCl cotransport) to the serosal medium induced significant increases in V b, FV a and high K +-induced ΔV b, indicating an increase in g K b. 4. 4. In the presence of serosal furosemide, Cl − removal from the serosal medium did not significantly alter V b, a Cl i or ΔV b from their corresponding values when serosal Cl − was present. 5. 5. Serosal furosemide had no significant effect on a K i and a Cl i measured with double-barreled ion-selective microelectrodes. 6. 6. These results suggest the possibility of a reciprocal relationship between g K b and the rate of basolateral KCl cotransport. This may contribute to the maintenace of a K i in gallbladder epithelial cells.

Electrical transients produced by the toad bladder in response to altered serosal composition at constant osmolality

The effects of step-changes in the ionic composition of the serosal medium bathing the toad urinary bladder under voltage-clamped conditions have been studied. A decrease in the K + concentration from 4 to 3 mmol/l in the serosal fluid increased transiently the transepitheliai current which after 30 min returned to the initial value. The peak current was reached after 3 min. The current response of the bladder to the reverse step in K + concentration, from 3 to 4 mmol/l was much smaller. Surprisingly, the partial replacement of Cl − with gluconate produced a transient increase in current. It is suggested that secondary active transport plays an important role in this phenomenon and leads to an increased apical Na + conductance. The second phases of the biphasic responses to Na/K + and Cl −/gluconate substitutions have been interpreted as osmotic effects. Since the exchange of solutions in these studies was isoosmotic but not necessarily isotonic, experiments were also performed with osmotic changes in the serosal fluid for the purpose of comparison.

The effects of indigestible dextrin on sugar tolerance: I. Studies on digestion-absorption and sugar tolerance

It is widely acknowledged that high viscosity water-soluble dietary fibers such as pectin and guar gum affect a lowering of blood glucose levels and a reducing of insulin secretion following a sugar load. However, as dietary fibers vary in origin and in chemical properties, their physiological functions differ as well. In this study the effects of Indigestible Dextrin (PF-C), a low viscosity, water-soluble dietary fiber obtained through acid and heat-treatment of potato starch, on various aspects of sugar tolerance were examined. First, the influence of PF-C on sucrose hydrolysis was examined in rat intestinal mucosa cell homogenate confirming that PF-C did not inhibit sucrase activity. Then, in order to investigate the influence of PF-C on sugar digestion-absorption, an experiment was performed by using the everted intestinal sac of the rat in vitro. PF-C did not have an effect on glucose-transport into the serosal medium, whereas PF-C did inhibit the transport of hydrolyzed-glucose from sucrose, with no change in the hydrolysis of sucrose. Recently, Crane et al. reported that there is a specific route for hydrolyzed glucose from sucrose in glucose-absorption on the enteric surface (disaccharidase related transport system). The possibility exists that PF-C specifically affects this pathway. Further, total glucagon released into the serosal medium stimulated by both glucose and sucrose were reduced by PF-C. On the basis of these results, an oral sugar tolerance test was conducted in both rats and healthy human subjects. In male Sprague-Dawley rats (8 weeks old, 250-280g) concurrent administration of PF-C (0.6g/kg body weight) reduced an increase in plasma insulin levels with no change in glucose levels following a glucose (1.5g/kg body weight) load. Further noted were reductions in increases in both plasma glucose and insulin levels following a sucrose (1.5g/kg body weight) plus PF-C (0.6g/kg body weight) load to that of the sucrose (1.5g/kg body weight) single load. These findings reflect the above mentioned in vitro results. Moreover, in healthy male subjects the increase in both plasma insulin and glucagon-like immunoreactivity (Gut GLI) levels following a Trelan-G75 load were significantly reduced by concurrent administration of PF-C. From these observations it would appear that the effectiveness of reducing insulin secretion by PF-C results due to the decrease in sugar absorption by inhibiting the disaccharidase-related transport system.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of potassium-free media and ouabain on epithelial cell composition in toad urinary bladder studied with X-ray microanalysis.

The technique of X-ray microanalysis was used to study the composition of toad urinary bladder epithelial cells incubated in Na Ringer's and K-free medium, with and without ouabain. Following incubation under short-circuit conditions, portions of tissue were coated with an external albumin standard and plunge-frozen. Cryosections were freeze-dried and analyzed. In Na Ringer's, granular and basal cells, and also the basal portion of the goblet cells, had similar water and ion compositions. In contrast, mitochondria-rich cells contained less Cl and Na. On average, the granular cells and a subpopulation of the basal cells lost K and gained Na after ouabain and in K-free medium alone. However, there was considerable variation from cell to cell in the responses, indicating differences between cells in the availabilities of ion pathways, either as a consequence of differences in the numbers of such pathways or in their control. In contrast, the compositions of both the low Cl, mitochondria-rich cells and a sub-population of the basal cells were little affected by the different incubation conditions. This is consistent with a comparatively low Na permeability of these cells. The results also indicate that (i) much, if not all, of the K in the dominant cell type, the granular cells, is potentially exchangeable with serosal medium Na, and (ii) Na is accumulated from the serosal medium under K-free conditions. They also provide information about the role of the (Na-K)-ATPase in the maintenance of cellular K in K-free medium, being consistent with other evidence that removal of serosal medium K inhibits transepithelial Na transport by decreasing Na entry to the cells from the mucosal medium, rather than solely by inhibiting the basolateral membrane (Na-K)-ATPase.

Application of membrane potential equations to tight epithelia

It is shown that equations developed to analyze the contributions of secondary active transport processes to symmetrical cells (Gordon, L.G.M., Macknight, A.D.C., 1991, J. Membrane Biol. 120:139-152) can be used, with minor modifications, to analyze the steady-state membrane potential in epithelia under the unique situation of short circuiting. Only under such conditions is there a single intracellular potential relative to both the mucosal and serosal media. The equations are investigated in relation to a model tight epithelium--the toad urinary bladder. It is shown that the properties of the membrane transport pathways are such that the intracellular potential under short-circuit conditions must be more negative than often reported. Given measurements of membrane potential and of voltage-divider ratio, it is possible to use the equations to estimate the absolute values of the membrane permeabilities and conductances under short-circuit conditions.

Lithium absorption by the rabbit gall‐bladder

Lithium (Li+) absorption across the low-resistance epithelium of the rabbit gall-bladder was studied in order to elucidate possible routes and mechanisms of Li+ transfer. Li+ at a concentration of 0.4 mM in both mucosal and serosal media did not affect isosmotic mucosa-to-serosa fluid absorption. At this low concentration net mucosa-to-serosa Li+ absorption was insignificant when the ambient Na+ concentration was 115 mM, although the gall-bladder had a significant Li+ permeability (2.7 X 10(-5) cm s-1) and a significant mucosa-to-serosa Li+ gradient developed as a result of fluid absorption. Net Li+ absorption was induced at reduced mucosal Na+ concentrations (by lowering the Na+ concentration down to 50 mM with or without substitution with sucrose, or by adding sucrose to the mucosal medium). This Li+ absorption occurred even in the absence of a mucosa-to-serosa Li+ gradient. Na+ and Li+ absorptions occurring at 50 mM Na+ were inhibited to the same degree by mucosal 1 mM amiloride. Substitution of 5-50 mM (44%) Na+ by Li+ in the external medium dose-dependently depressed Na+ absorption by up to 76%, while substitution by 50 mM choline had no significant effect. Li+ inhibition of Na+ absorption was elicited from the mucosal side and was not accounted for by compensatory Li+ absorption; water and Na+ absorption rates decreased nearly in parallel. The effects of 0.4 mM amiloride and of substitution with 20 mM Li+ were only partly additive. It is concluded that Li+ absorption in the rabbit gall-bladder cannot be explained by passive (paracellular) transport, but must be the result of transcellular, active transport. Both at low and at high concentrations Li+ may enter the cell via an Na+/H+ exchanger in the apical cell membrane. At high concentrations Li+ may inhibit Na+ absorption by interference with the exchange mechanism and/or via effects at the cytoplasmic level. The Li+ transfer mechanism across the basolateral cell membrane remains unknown.

Amiloride inhibits the vasopressin-induced increase in epithelial water permeability

The vasopressin (VP)-induced increase in water permeability in high-resistance, amphibian epithelia is not altered by the abolition of net Na+ flux caused by amiloride added to the apical bathing medium. In this work we looked at the effects on water transport of amiloride added to the serosal medium at a concentration (10(-3) M) known to inhibit Na+/H+ exchange. In urinary bladders of Bufo marinus, amiloride partially blocked the hydrosmotic response to VP. A similar inhibition was found with cyclic adenosine 5'-monophosphate (cAMP) or serosal hypertonicity. We hypothesized that this effect of amiloride could be due to an inhibition of Na+/H+ and/or Na+/Ca2+ antiporters present in the epithelial basolateral membrane and looked at the effects of the diuretic in Na(+)-free media. A similar degree of inhibition of water flow was still found, thus showing that amiloride acts on a cell target other than the antiporters. In toad skin, amiloride did not inhibit the hydrosmotic response to VP and to isoproterenol; however the response to high K+ was significantly reduced. Among the amiloride cell targets described so far, adenylate cyclase and protein kinase A appear to be the best candidates to explain the inhibition of the hydrosmotic response reported here. Direct measurements of intracellular cAMP are needed however to substantiate this hypothesis.