Transapical Na+ electrochemical potential difference and solute accumulation in Aplysia californica enterocytes

Extracellular water averaged 20% of total intestinal water. The sugar, D-galactose, was accumulated by the Aplysia enterocytes. The amino acids glycine, L-alanine and alpha-aminoisobutyric acid (AIB) were all accumulated by the Aplysia enterocytes. The electrochemical potential difference for Na+ across the mucosal membrane of Aplysia enterocytes was energetically adequate in driving D-galactose, L-alanine, glycine or AIB uphill into the Aplysia enterocytes.     

Energetics of coupled Na+ and Cl- entry into epithelial cells of bullfrog small intestine.

Na+, K+ and Cl- concentrations (cij) and activities (aij), and mucosal membrane potentials (Em) were measured in epithelial cells of isolated bullfrog (Rana catesbeiana) small intestine. Segments of intestine were stripped of their external muscle layers, and bathed (at 25 degrees C and pH 7.2) in oxygenated Ringer solutions containing 105 mM Na+ and Cl- and 5.4 mM K+. Na+ and K+ concentrations were determined by atomic absorption spectrometry and Cl- concentrations by conductometric titration following extraction of the dried tissue with 0.1 M HNO3. 14C-labelled inulin was used to determine extracellular volume. Em was measured with conventional open tip microelectrodes, aiCl with solid-state Cl-selective silver microelectrodes and aiNa and aiK with Na+ and K+-selective liquid ion-exchanger microelectrodes. The average Em recorded was -34mV. ciNa, ciK and ciCl were 51, 105 and 52 mM. The corresponding values for aiNa, aiK and aiCl were 18, 80 and 33 mM. These results suggest that a large fraction of the cytoplasmic Na+ is 'bound' or sequestered in an osmotically inactive form, that all, or virtually all the cytoplasmic K+ behaves as if in free solution, and that there is probably some binding of cytoplasmic Cl-. aiCl significantly exceeds the level corresponding to electrochemical equilibrium across the mucosal and baso-lateral cell membranes. Earlier studies showed that coupled mucosal entry of Na+ and Cl- is implicated in intracellular Cl- accumulation in this tissue. This study permitted estimation of the steady-state transapical Na+ and Cl- electrochemical potential differences (deltamuNa and deltamuCl). deltamuNa (-7000 J . mol-1; cell minus mucosal medium) was energetically more than sufficient to account for deltamuCl (1000--2000 J . mol-1).     

Endocytosis and Na+/solute cotransport in renal epithelial cells

Endocytic uptake of [3H]sucrose and lucifer yellow, markers for fluid-phase endocytosis, was studied in cultures of the renal epithelial cell lines LLC-PK1 and OK. Endocytosis in LLC-PK1 cells was inhibited when the cells were grown in the presence of gentamicin (1 mg/ml) for 4 days or when the cells were treated with concanavalin A (1 mg/ml) for 5 h. These changes occurred without perturbation of intracellular Na+ and K+ content, indicating that the cells maintained normal ion gradients. The inhibition of endocytosis was accompanied by marked increases in the apparent Vmax for Na+-dependent cell uptake of solutes such as Pi and L-alanine. The apparent Km was unchanged. In contrast, treatment of OK cells with concanavalin A produced marked stimulation of endocytosis and inhibition of the Na+-dependent uptake of Pi and L-glutamate. These changes occurred in the absence of changes in intracellular Na+ and K+ content. Neither gentamicin nor concanavalin A had a direct effect on Na+/solute cotransport in these cell lines. The changes in Na+/Pi cotransport induced by concanavalin A in both LLC-PK1 and OK cells were blocked by keeping the cells at 4 degrees C during exposure to the lectin, suggesting that endocytosis may be part of the mechanism which mediates the changes in solute uptake. The reciprocal relationship between the changes in endocytosis and the changes in Na+/solute cotransport is consistent with the possibility that the number of Na+/solute cotransporters present in the plasma membrane may be altered by an increase or decrease in the rate of membrane internalization by endocytosis. The Vmax changes in Na+/solute cotransport provide indirect support for this conclusion.     

α-Adrenergic inhibition of cyclic AMP accumulation in epithelial cells isolated from rat small intestine

The present work shows that α-adrenergic agonists induce the suppression of basal and hormone-stimulated cyclic AMP levels in rat intestinal epithelial cells. Epinephrine (100 μM) suppresses by 35% the cyclic AMP levels evoked by the vasoactive intestinal peptide (VIP). The adrenergic agent induces a similar percentage of inhibition at 15, 30 and 37°C. Addition of epinephrine 20 min prior to, on 5 or 20 min after VIP yields the same magnitude of inhibition as when performed together with the stimulus. The α-adrenergic agent does not alter the K_0.5 of VIP in stimulating cyclic AMP production but reduces its efficacy. Epinephrine also suppresses prostaglandin E₁- and E₂-stimulated cyclic AMP levels by about 35%. The lowest effective concentration of epinephrine required to suppress VIP-stimulated cyclic AMP levels is 0.1 μM, half-maximal (K_0.5) and maximal effects being observed at 5 and 100 μM, respectively. Norepinephrine has the same efficacy but a slightly lower potency (K_0.5 = 18 μM) than epinephrine. Phenylephrine acts as a partial agonist of very low potency; clonidine has very little intrinsic activity and antagonizes the inhibition by epinephrine. The inhibition of VIP-stimulated cyclic AMP levels is observed in the absence of any blocking agents. It is not affected by the β blocker propranolol, but is completely reversed with α blockers with the following order of potency: dihydroergotamine>yohimbine>phentolamine. Yohimbine is much more potent than prazosin, which only partially reverses the inhibition induced by epinephrine. It is concluded that α-adrenoreceptors of the α₂ subtype mediate the suppression of VIP-stimulated cyclic AMP levels in intestinal epithelial cells. This effect is likely to be due to the inhibition of adenylate cyclase within intact cells as epinephrine is able to reduce adenylate cyclase activity of intestinal epithelial cell plasma membranes.     

Lipoproteins of rat small intestine epithelial cells in D-hypovitaminosis

Chemical composition of lipoproteins has been studied in intestinal epitheliocytes of rats in normalcy and under D-hypovitaminosis. It is found that D-hypovitaminosis induces changes in the lipid and protein composition of lipoproteins. It is supposed that disturbances in biosynthesis of the lipoprotein components and their transport may be possible reasons of such changes.     

Catalase particles in the epithelial cells of the guinea-pig small intestine

Synopsis Purified preparations of epithelial cells have been made from the guinea-pig small intestine. Homogenates of these preparations have been analysed by centrifugation in a zonal rotor. The results confirm the presence of lysosomes in these cells and indicate the existence of catalase particles which equilibrate in a sucrose gradient at a density of between 1.21 and 1.23 and which have a different distribution from other subcellular particles except lysosomes. Injection of Triton WR-1339 into fasting animals enables the separation of lysosomes and catalase particles.     

Protein kinase C from small intestine epithelial cells

Protein kinase C activity has been identified in cytosolic and membrane fractions from rat and rabbit small intestine epithelial cells. The cytosolic fraction comprised about the 75% of total activity. Protein kinase C activity was resolved from other protein kinase activities by ion exchange chromatography. Phosphatidylserine or phosphatidylinositol were required for protein kinase C to be active. In addition, the activity was enhanced by the presence of a diacylglycerol. Diolein and dimyristin were the most effective (13-14 fold activation). In the presence of phosphatidylserine and diolein, the Ka for activation by Ca2+ was 10(-7)M. The phorbol ester TPA substituted for diacylglycerol in activating protein kinase C. Brush border and basolateral membranes contained protein kinase C activity, although the specific activity of the basal lateral membranes was four-fold higher than the specific activity of the brush border membranes. The presence of PKC in small intestine epithelial cells might have important implications in the Ca2+ mediated control of ionic transport in this tissue.     

L-MBP is expressed in epithelial cells of mouse small intestine

The mannan-binding proteins (L-MBP and S-MBP, also denoted MBL-C and MBL-A), mainly produced in liver and existing in liver and serum, play important roles in the innate immunity against a variety of pathogens. Total RNA from mouse tissues were screened for MBP mRNA by RT-PCR. In addition to liver, S-MBP mRNA was detected in lung, kidney, and testis, and L-MBP mRNA was detected in kidney, thymus, and small intestine. Quantitative RT-PCR revealed that the small intestine is a predominant site of extrahepatic expression of L-MBP. Western blotting with polyclonal Abs against rat L-MBP demonstrated this protein in Triton X-100 extracts of the small intestine obtained from mice that had undergone systemic perfusion. Immunohistochemical staining with an mAb against mouse L-MBP and in situ hybridization revealed that L-MBP is selectively expressed in some villous epithelial cells of the small intestine. These findings suggest that L-MBP plays a role in mucosal innate immunity.     

Transmucosal passage of polyalkylcyanoacrylate nanocapsules as a new drug carrier in the small intestine

The enteral absorption of particles has been investigated in the dog using a colloidal drug carrier, polyalkylcyanoacrylate nanocapsules loaded with an iodized oil (Lipiodol), as a tracer for X-ray microprobe analysis in a scanning electron microscope. Nanocapsules are spherical capsules, 100 to 200 nm in diameter, with a continuous polymeric wall surrounding a cavity which encapsulates the drug. Administered in the jejunal lumen, Lipiodol nanocapsules improved the absorption of the tracer as indicated by increased concentration of iodine in the plasma of mesenteric blood. In order to follow nanocapsules at the cellular level, all tissue compartments were preserved in a life-like state by cryofixation and freeze-drying of intestinal biopsies. Nanocapsules appeared in the intestinal lumen close to the mucus, then in intercellular spaces and defects of the mucosa and finally in the lamina propria and blood capillaries; in this latter compartment, the iodine content was four-fold higher than after intra-jejunal administration of Lipiodol emulsion. This complete phenomenon occurred only at the tip of the villi and happened within less than 60 min. We conclude that nanocapsules enhance the rate of absorption of Lipiodol and transport the drug from the intestinal lumen to the vascular compartment using a paracellular pathway. Thus they may be useful as drug carrier for oral administration of many chemicals.     

Methodical aspects of obtaining isolated small intestine epithelial cells

Based on the data from literature and of the author's results, the methods for isolation of small intestine epithelial cells have been analyzed by the following criterion: object of investigation, procedures for cell isolation--mechanical, chemical, enzymatic, and biochemical properties.     

Na+-stimulated ATPase activities in basolateral plasma membranes from guinea-pig small intestinal epithelial cells

Two ATPase activities, a Na+-ATPase and a (Na+ + K+)-ATPase, have been found associated with sheets of basolateral plasma membranes from guinea-pig small intestinal epithelial cells. The specific activity of the former is 10-15% of the latter. The two ATPase activities differ in their affinity for Na+, their optimal pH, their K+ requirement and particularly in their behaviour in the presence of some inhibitors and of Ca2+. Thus the Na+-ATPase is refractory to ouabain but it is strongly inhibited by ethacrynic acid and furosemide, whilst the (Na+ + K+)-ATPase is totally suppressed by ouabain, partially by ethacrynic acid and refractory to furosemide. In addition, the Na+-ATPase is activated by micromolar concentrations of calcium and by resuspension of the membrane preparation at pH 7.8. The Na+-ATPase is only stimulated by sodium and to a lesser extent by lithium; however, this stimulation is independent of the anion accompanying Na+. The latter rules out the participation of an anionic ATPase. The relation between the characteristics of the sodium transport mechanism in basolateral membrane vesicles (Del Castillo, J.R. and Robinson, J.W.L. (1983) Experientia 39,631) and those of the two ATPase activities present in the same membranes, allow us to postulate the existence of two separate sodium pumps in this membranes. Each pump would derive the necessary energy for active ion transport from the hydrolysis of ATP, catalyzed by different ATPase systems.     

Phosphodiesterase II in epithelial cells from guinea-pig and rat small intestine

Phosphodiesterase II activity was determined by using a synthetic substrate, the 2,4-dinitrophenyl ester of thymidine 3'-phosphate. The enzyme activity was determined in fractions obtained by differential centrifugation of homogenates of epithelial cells from the small intestinal mucosa of guinea pigs and rats. In guinea-pig preparations phosphodiesterase II occurred with highest specific activity in those fractions rich in succinate dehydrogenase and acid phosphatase. A lysosomal location for the guinea-pig enzyme was indicated by its structure-linked latency and by its association with particles that under-went a characteristic decrease in equilibrium density when Triton WR-1339 was injected into the animals. With rat preparations a much greater proportion of the phosphodiesterase II activity was found in the soluble fraction after ultracentrifugation. The rat enzyme exhibited a lower degree of latency and administration of Triton WR-1339 had no effect. The rat enzyme further differed from that of the guinea pig in other respects; it was more labile at 60 degrees C, it exhibited a lower pH optimum and it had a higher molecular weight as determined by gel-filtration chromatography.     

Phosphofructokinase D from the epithelial cells of rat small intestine.

Phosphofructokinase from the epithelial cells of rat small intestine was characterized with respect to isoenzyme type in a comparison of its properties with those of the skeletal-muscle, brain and major liver isoenzymes by using five different techniques, namely electrophoresis on cellulose acetate and in polyacrylamide gels, chromatography on DEAE-cellulose, (NH4)2SO4 precipitation and immunotitration. When precautions were taken to inhibit the formation of active proteolytic artifacts by the action of endogenous proteinases, each technique revealed that rat intestinal mucosa contains only a single form of phosphofructokinase. The mucosal isoenzyme was found to be very similar to, although not identical with, the major liver isoenzyme and to be quite distinct from the skeletal-muscle isoenzyme when studied by the techniques of cellulose acetate electrophoresis, chromatography on DEAE-cellulose and immunotitration, whereas the converse was true when studied by the techniques of (NH4)2SO4 precipitation and polyacrylamide-gel electrophoresis. The mucosal isoenzyme was distinct from the brain isoenzyme when studied by each of the five techniques. Tsai & Kemp [(1973) J. Biol. Chem. 248, 785-792] reported that animal tissues contain three principal isoenzymes of phosphofructokinase, type A found as the sole isoenzyme in skeletal muscle, type B found as the major isoenzyme in liver and type C found as a significant isoenzyme in brain. Phosphofructokinase from mucosa is distinct from each of these isoenzymes. Following the nomenclature of Tsai & Kemp (1973), the isoenzyme from the mucosa of rat intestinal epithelial cells is designated phosphofructokinase D. The mucosal and liver isoenzymes behave so similarly with respect to their charge and immunological characteristics, on which the typing of isoenzymes is conventionally based, that it is likely that some tissues reported to contain the liver isoenzyme contain instead the mucosal isoenzyme.     

Structural and compositional difference in the neutral glycolipids between epithelial and non-epithelial tissue of the mouse small intestine

Five major neutral glycolipids, GL-1-GL-5, were isolated from the the mouse small intestine. Their structures and distribution were determined by permethylation analysis, sequential degradation with exoglycosidases and/or immunohistochemistry. The molar ratio of GL-1, GL-2, GL-3, GL-4 and Gl-5 in the whole small intestine was 1:0.04:0.03:0.42:0.02. The structures of GL-1 and GL-4 present in epithelial cells were reported previously to be glucosyl ceramide and asialo G_M1, respectively (Umesaki, Y., Suzuki, A., Kasama, T., Tohyama, K., Mutai, M. and Yamakawa, T. (1981) J. Biochem. 90, 1731–1738). GL-5, also present in the epithelial cells, was fucosyl asialo G_M1, and fucose was shown to be linked to terminal galactose of asialo G_M1 in the manner of α(1–2) bond. GL-2 and GL-3, present in the residual tissue after scraping the mucosa, were determined to be globoside and Forssman glycolipid, respectively. Both globoside and Forssman glycolipid of the non-epithelial tissue had non-hydroxy fatty acid (C₁₆–C₂₄) in combination with sphingosine (C₁₈) as the ceramide components, in contrast with the ceramide structures of the epithelial glycolipids, which contained α-hydroxy fatty acids in combination with phytosphingosine. Immunohistochemical staining using anti-glycolipid antibodies confirmed the distribution of asialo G_M1 and fucosyl asialo G_M1, and Forssman glycolipid in the epithelial and non-epithelial tissue, respectively.