Role of villus microcirculation in intestinal absorption of glucose: coupling of epithelial with endothelial transport.

Capillaries in jejunal villi can absorb nutrients at rates several hundred times greater (per gram tissue) than capillaries in other tissues, including contracting skeletal muscle and brain. We here present an integrative hypothesis to account for these exceptionally large trans-endothelial fluxes and their relation to epithelial transport. Equations are developed for estimating concentration gradients of glucose across villus capillary walls, along paracellular channels and across subjunctional lateral membranes of absorptive cells. High concentrations of glucose discharged across lateral membranes to subjunctional intercellular spaces are delivered to abluminal surfaces of villus capillaries by convection-diffusion in intercellular channels without significant loss of concentration. Post-junctional paracellular transport thus provides the series link between epithelial and endothelial transport and makes possible the large trans-endothelial concentration gradients required for absorption to blood. Our analysis demonstrates that increases of villus capillary blood flow and permeability-surface area product (PS) are essential components of absorptive mechanisms: epithelial transport of normal digestive loads could not be sustained without concomitant increases in capillary blood flow and PS. The low rates of intestinal absorption found in anaesthetised animals may be attributed to inhibition of normal villus microvascular responses to epithelial transport.

Role of pre-epithelial "unstirred" layers in absorption of nutrients from the human jejunum.

Pre-epithelial "unstirred" layers (abbr. pL) are generally regarded as undesirable diffusion barriers that impede access to absorptive cells of exogenous hexoses, amino acids or other experimental probes added to fluids bathing the mucosal surface. In the present paper it is suggested that the pL may have a functional role. Diffusion plus convection of saccharides and oligopeptides from lumen to brush border, combined with absorption to blood of their hydrolytic products, confers rectifying properties to the pL. The proposed model, based on experimental data from segmental jejunal perfusions in normal human subjects, indicates that the functional pathway for diffusion plus convection through the pL of hexoses and amino acids bound in the form of oligomers is only 10 +/- 2 microm or little more than the anatomical thickness of the glycocalyx and mucus layers. In contrast, the pathlength from brush border to lumenal perfusion fluid for diffusion minus convection of monomers generated by membrane bound hydrolases is 50-150 microm. According to this model the pL offers little resistance to the passage of saccharides or oligopeptides from lumen to brush border but at the same time it provides a protective blanket that diminishes diffusional losses to lumenal chyme of hexoses and amino acids generated in the brush border. The model provides a theoretical explanation for the "kinetic advantage" of transporting hexoses or amino acids through the pL in the form of oligomers and it predicts the proximal-distal concentrations of free glucose or fructose found experimentally in the outflows from jejunal segments perfused with sucrose or maltose.

Scaling of dimensions of small intestines in non-ruminant eutherian mammals and its significance for absorptive mechanisms.

The mucosal surface area of small intestines in non-ruminant eutherian mammals increases approximately in proportion to the 0.6 power of body mass, whereas resting metabolic rate (RMR) increases approximately with the 0.74 power of body mass; the mass exponent for field metabolic rates (FMR) may exceed 0.8. These relationships imply that the average rate of absorption of metabolic substrates, expressed per unit area of mucosal surface, is greater in large animals than in small. In the present paper I collate data from the literature relating mucosal surface area, fluid absorption and glucose transport rates to body size. Glucose-stimulated fluid absorption per unit area of mucosal surface increases with body size, whereas transcellular, carrier-mediated glucose transport per unit area decreases with body size. In perfused jejunal segments of normal human subjects the rates of fluid absorption per unit area of mucosa are five to ten times greater than in laboratory rats. The absorbed fluid contains glucose in amounts that may greatly exceed the maximum transport capacity of the apical glucose transporter. It follows that the paracellular component of glucose absorption increases with body size. Scaling of intestinal dimensions and transport therefore provides new information about the relative contributions of transcellular and paracellular pathways to absorption of nutrients.

Absorption and excretion of undegradable peptides: role of lipid solubility and net charge.

Absorption and excretion of undegradable peptides were investigated with use of octapeptides synthesized from D-amino acids. D-Tyrosine was included in each peptide to permit labeling with 125I, D-glutamic acid or D-lysine were included to vary net electric charge and D-serine or D-leucine were included to vary lipid solubility. Peptides were administered parenterally or orally to normal rats drinking 5% glucose or maltose. Forty-five percent of a lipid-insoluble, negatively charged octapeptide added to the drinking fluid in milligram quantities was absorbed from the intestine and excreted intact in urine; 90% of this peptide was recovered in urine after parenteral injection. In contrast, lipophilic D-octapeptides were largely excreted in feces, even after subcutaneous injection; the amounts excreted in feces were correlated with oil/aqueous partition coefficients. Evidence is presented that lipophilic peptides entering liver cells combine with bile salts to form hydrophilic complexes that are secreted rapidly at high concentration in bile. At physiological concentrations of bile salts (5-40 mM) and nanomolar concentrations of peptide the binding is so complete that these undegradable peptides are rapidly cleared from liver to duodenal fluid in association with the bile salts. After reaching the ileum the bile salts are reabsorbed to blood, leaving the original lipophilic peptides to be excreted in the feces from which they can be extracted, purified and identified by high-pressure liquid chromatography. These mechanisms are discussed in relation to a) the paracellular absorption of peptides and other solutes by solvent drag and b) the delivery and fate of biologically active peptides.

Intestinal absorption and excretion of octapeptides composed of D amino acids.

Octapeptides synthesized from D amino acids were absorbed from the intestine and excreted in urine of normal rats drinking 5% glucose/1% creatinine containing the 125I-labeled peptides at 0.1-25 mg/dl. The rats ingested fluid at the rate of about 20 ml/hr and produced urine at 15 ml/hr for several hours during the nocturnal feeding period. Sixty-one +/- 4% of the ingested creatinine and 50 +/- 3% of a lipid-insoluble D octapeptide (EASASYSA, 784 Da) were excreted intact in the urine. The steady-state molar rate of absorption-excretion of creatinine equaled or exceeded the maximum rate of carrier-mediated intestinal transport of glucose, suggesting that both the creatinine and the D octapeptide were transported paracellularly by solvent drag through absorptive cell junctions that were dilated by the glucose. More than 70% of the ingested glucose was also absorbed paracellularly. The results demonstrate that intact oligopeptides can be absorbed efficiently from the intestine when they are not hydrolyzed by membrane-bound peptidases of the brush border. The results also provide support for recent theories proposing that coupling of membrane digestion with paracellular solvent drag accounts for a major fraction of normal intestinal absorption of nutrients.

On the coupling of membrane digestion with intestinal absorption of sugars and amino acids.

An hypothesis is advanced to account for the large paracellular component of absorption of nutrients by the small intestine. High concentrations of hexoses and amino acids in the immediate vicinity of transporters and cell junctions are generated by membrane-bound saccharases and peptidases. After saturation of membrane carriers, the concentrations of monomers in the microenvironment increase until paracellular plus transcellular absorption equals rates of formation. During hydrolysis of maltose, the concentration of glucose at which rates of absorption and formation are equal is 200-300 mM, and at these concentrations paracellular transport accounts for 60-80% of total absorption. Transcellular concentrative transport provides the force for osmotic flow and at the same time triggers contraction of perijunctional actomyosin, thereby increasing transjunctional coefficients of osmotic flow and solvent drag. Paracellular absorption requires no oxidative energy other than that required to maintain the transjunctional concentration gradient and to energize contraction of cytoskeletal elements controlling junctional permeability and functional surface of lateral membranes. Almost all glucose generated by membrane hydrolysis is absorbed, but some may diffuse from high concentration in the microenvironment to the lumen thus accounting for the small amounts of free glucose found in macrosamples of chyme. Unstirred layers adjacent to the brush border retard backdiffusion to lumen, thus maintaining high concentrations for passive paracellular absorption. Coupling of membrane digestion with paracellular transport provides almost perfect matching of absorption to digestive loads because transport by solvent drag is proportional to concentration at cell junctions and this in turn is proportional to rate of hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Transmucosal impedance of small intestine: correlation with transport of sugars and amino acids.

Transmucosal impedances of isolated perfused segments of jejunum from mice and hamsters were measured at frequencies from 10-100,000 Hz in the presence and absence of sugars and amino acids. Na-coupled transport of organic substrates caused large decreases of transmucosal impedance, reflecting contraction of cytoskeletal proteins controlling permeability of tight junctions, functional surface of basolateral membranes, and width of extracellular pathways. The observed changes of impedance were closely correlated with molar rates of Na-coupled active transport rather than with molecular species. Thus amino acids and sugars having the same molar rates of active transport also have the same effects on transmucosal impedance. It is proposed that a nonspecific increase of intracellular osmotic pressure during active transport is the first step initiating cytoskeletal contraction. Cell volume regulatory responses, including increased basolateral K+ conductance and Ca2+ influx, may be subsequent steps leading to contraction of perijunctional actomyosin, formation of junctional dilatations, and exposure of lateral membranes. Enhancement of oxygen capacity of perfusion fluids (e.g., with fluorocarbon emulsion) is required to maintain viability of isolated intestinal epithelium; in plain oxygenated Ringer-HCO3 solution, the transmucosal impedance is abnormally low and cytoskeletal contractile responses to Na-coupled transport are attenuated. An electrical circuit analog is presented that simulates almost exactly the observed transmucosal impedances and provides quantitative evaluation of the effects of Na-coupled transport of sugars and amino acids on resistances of tight junctions, capacitance of basolateral membranes, and postjunctional resistances of lateral intercellular spaces and villus cores.

Paracellular intestinal absorption of glucose, creatinine, and mannitol in normal animals: relation to body size.

Mice, rats, or rabbits were provided with a liquid diet of 10-12% glucose (Glc), 0.5-1% creatinine, 1-2% mannitol, and mannitol labeled with 3H on a terminal carbon. Average rates of ingestion of Glc were greater than maximum rates of active, carrier-mediated Glc transport reported for the intestines of these species. The discrepancy was small in mice but increased exponentially with body weight (BW). Ingestion-absorption of Glc increased exponentially with the 0.73 power of BW as expected from metabolic rate, whereas active transport, estimated from the literature, varied exponentially with the 0.50 power of BW. It is estimated that in humans the ingestion-absorption rate of Glc may be 10-20 times greater than active transport. In the presence of Glc, 50-65% of the ingested creatinine was recovered in urine compared with 75-85% recovered after intraperitoneal or subcutaneous injections. The amount of creatinine recovered in urine depended on the amount of ingested Glc, as predicted from the effects of Glc on width and permeability of absorptive cell junctions. Eighty percent or more of the 3H label on mannitol was recovered in urine or other body fluids, although most of the [3H]mannitol was oxidized to [3H]water after being absorbed intact from the intestine. It is concluded that in the presence of Glc, creatinine and mannitol (together with Glc, amino acids, and other small nutrients) are absorbed passively by solvent drag between absorptive cells, as found previously in anesthetized rats (J. R. Pappenheimer and K. Z. Reiss. J. Membr. Biol. 100: 123-136, 1987). The ratio of solvent drag to carrier-mediated transport increases exponentially with BW and may account for the capacity of human intestines to absorb large amounts of Glc during prolonged exercise, Glc tolerance tests, or oral Glc-saline therapy for dehydration.

Physiological regulation of epithelial junctions in intestinal epithelia.

This symposium paper is a digest of three full-length manuscripts currently in press with J Membrane Biology (see reference list). The three papers provide evidence that sugars, amino-acids and small peptides are transported through intestinal epithelium primarily by solvent drag through paracellular channels. Active transport of sugars and amino acids plays a necessary but nevertheless secondary role in the mass transport from intestinal lumen to blood at physiological concentrations. Na-coupled solute transport serves two principal functions - a) it inserts relatively small amounts of solutes at high concentration into the intercellular spaces below the occluding junctions thereby providing the force for osmotic flow and solvent drag; b) it triggers contraction of the perijunctional actomyosin ring, thereby widening the occluding junctions and providing optimal conditions for transport of luminal nutrients in bulk by solvent drag. Active transport of glucose reaches its maximum capacity (V max) at luminal concentrations of 10-15 mM whereas transport by solvent drag increases in proportion to luminal concentration; at concentrations normally present in the duodenum and upper jejunum after a meal (50-300 mM) transport through paracellular spaces by solvent drag accounts for 60-90% of total glucose absorbed into blood. Similar considerations apply to other hydrophilic nutrients including amino acids, small saccharides and peptides. As nutrients are removed from the upper intestine by the above mechanisms, their concentrations decrease and the "traditional" role of active transport becomes a greater fraction of total absorption.(ABSTRACT TRUNCATED AT 250 WORDS)

Contribution of solvent drag through intercellular junctions to absorption of nutrients by the small intestine of the rat.

The lumen of the small intestine in anesthetized rats was recirculated with 50 ml perfusion fluid containing normal salts, 25 mM glucose and low concentrations of hydrophilic solutes ranging in size from creatinine (mol wt 113) to Inulin (mol wt 5500). Ferrocyanide, a nontoxic, quadrupally charged anion was not absorbed; it could therefore be used as an osmotically active solute with reflection coefficient of 1.0 to adjust rates of fluid absorption, Jv, and to measure the coefficient of osmotic flow, Lp. The clearances from the perfusion fluid of all other test solutes were approximately proportional to Jv. From Lp and rates of clearances as a function of Jv and molecular size we estimate (a) the fraction of fluid absorption which passes paracellularly (approx. 50%), (b) coefficients of solvent drag of various solutes within intercellular junctions, (c) the equivalent pore radius of intercellular junctions (50 A) and their cross sectional area per unit path length (4.3 cm per cm length of intestine). Glucose absorption also varied as a function of Jv. From this relationship and the clearances of inert markers we calculate the rate of active transport of glucose, the amount of glucose carried paracellularly by solvent drag or back-diffusion at any given Jv and luminal glucose concentration and the concentration of glucose in the absorbate. The results indicate that solvent drag through paracellular channels is the principal route for intestinal transport of glucose or amino acids at physiological rates of fluid absorption and concentration. In the absence of luminal glucose the rate of fluid absorption and the clearances of all inert hydrophilic solutes were greatly reduced. It is proposed that Na-coupled transport of organic solutes from lumen to intercellular spaces provides the principal osmotic force for fluid absorption and triggers widening of intercellular junctions, thus promoting bulk absorption of nutrients by solvent drag. Further evidence for regulation of channel width is provided in accompanying papers on changes in electrical impedance and ultrastructure of junctions during Na-coupled solute transport.

Physiological regulation of transepithelial impedance in the intestinal mucosa of rats and hamsters.

Isolated intestinal segments from rats or hamsters were recirculated with balanced salt solutions containing fluorocarbon emulsion to provide 6 vpc oxygen. The lumen contained an axial Ag-AgCl electrode, and the serosal surface was surrounded by a cylindrical shell of Ag-AgCl. Transmural impedances were measured at frequencies from 0.01-30 kHz before and after removal of the mucosal epithelium. The resistance of intercellular junctions, RJ, the distributed resistance of the lateral spaces, RL, and the distributed membrane capacitance, CM, were computed from the relations between frequency and impedance. Activation of Na-coupled solute transport by addition of glucose, 3-0-methyl glucose, alanine or leucine caused two- to threefold decreases of transepithelial impedance. Typical changes induced by glucose in hamster small intestine were RJ 30----13 omega, RL 23----10 omega, and CM 8----20 microF (per cm length of segment). Half maximal response occurred at a glucose concentration of 2-3 mM. The area per unit path length of the junctions (Ap/delta chi = specific resistance divided by RJ) in glucose activated epithelium was 3.7 cm in hamster midgut and 6.8 cm in rat. These values are close to the 4.3 cm estimated independently from coefficients of solvent drag and hydrodynamic conductance in glucose-activated rat intestine in vivo. The transepithelial impedance response to Na-coupled solute transport was reversibly dependent upon oxygen tension. It is proposed that activation of Na-coupled solute transport triggers contraction of circumferential actomyosin fibers in the terminal web of the microvillar cytoskeletal system, thereby pulling apart junctions and allowing paracellular absorption of nutrients by solvent drag as described in the previous accompanying paper. Anatomical evidence in support of this hypothesis is presented in the following second accompanying paper.

Structural basis for physiological regulation of paracellular pathways in intestinal epithelia.

Isolated segments of hamster small intestine were perfused with oxygenated salt-fluorocarbon emulsions with or without 10-25 mM glucose, alanine or leucine. Resistances of intercellular occluding junctions and of lateral spaces and the distributed capacitance of epithelial plasma membranes were estimated from steady-state transepithelial impedances at frequencies from 0.01-10 kHz. The segments were then fixed in situ with isorheic 2.5% glutaraldehyde while continuing to measure impedance. This method of fixation increased the resistance of lateral spaces but had little effect on the resistance of occluding junctions or on membrane capacitance. The large decreases of impedance induced by glucose or amino acids were preserved in fixed tissue and could therefore be correlated with changes in structure. The observed changes of impedance were interpreted as decreased resistance of occluding junctions and lateral spaces together with increased exposed surface of lateral membranes (capacitance). Glucose, alanine or leucine induced expansion of lateral intercellular spaces as seen by light and electron microscopy. Large dilatations within absorptive cell occluding junctions were revealed by electron microscopy. Freeze-fracture analysis revealed that these dilatations consisted of expansions of compartments bounded by strands/grooves. These solute-induced structural alterations were also associated with condensation of microfilaments in the zone of the perijunctional actomyosin ring, typical of enhanced ring tension. Similar anatomical changes were found in epithelia fixed in situ at 38 degrees C during luminal perfusion with glucose in blood-circulated intestinal segments of anesthetized animals. These structural changes support the hypothesis that Na-coupled solute transport triggers contraction of perijunctional actomyosin, thereby increasing junctional permeability and enhancing absorption of nutrients by solvent drag as described in the two accompanying papers.

Hypoxic insomnia: effects of carbon monoxide and acclimatization.

Hypoxia causes severe disruption of both rapid-eye-movement (REM) and non-REM (NREM) sleep. Experiments were performed on rats to determine if hypoxic insomnia is mediated by peripheral chemoreceptors and if normal sleep is restored during acclimatization to low O2. Novel methods were devised to measure distribution of amplitudes of cortical slow waves during NREM sleep and to detect REM sleep from the ratio of amplitudes of theta-to delta-frequency bands in the hippocampal electroencephalogram (EEG). Acute exposure of rats to 10.5% O2 (5,030 m altitude equivalent) during daylight hours virtually abolished REM sleep and shifted the distribution of amplitudes of slow-wave sleep EEG toward awake values. Similar disruption of sleep occurred during inhalation of 0.05% CO with steady-state carboxyhemoglobin of approximately 35%. Respiratory rate and alveolar ventilation were greatly increased by 10.5% O2 but were unaffected by CO. Therefore, hypoxic disruption of sleep was not mediated by peripheral chemoreceptors regulating breathing. Partial recovery of sleep occurred after 1-2 wk of hypoxia, but both REM and NREM were still subnormal after 1 mo. Decreased intensity of NREM sleep during hypoxia, measured by amplitude of cortical slow waves, may explain the disparity between subjective complaints of insomnia at altitude and evaluations of sleep by direct observation or by conventional EEG. Loss of appetite, loss of weight, irritability, and other symptoms of altitude sickness may be related to hypoxic insomnia.

Peptidoglycans as promoters of slow-wave sleep. I. Structure of the sleep-promoting factor isolated from human urine.

Fast atom bombardment-mass spectrometry (FABMS) has been used to determine the structure of the urinary sleep-promoting factor (FSu), the nature of whose components had been reported earlier. Less than 1 nmol of the underivatized substance sufficed for the FABMS experiments. The major somnogenic constituent of the purified preparation was a peptidoglycan of Mr = 921 with the structure N-acetylglucosaminyl-N -acetylanhydromuramylalanylglutamyldiaminopimelylalanine. The anhydro linkage is between C-1 and C-6 of the muramyl entity. Two additional substances accompanied the above compound. These were the hydrated form (i.e. in which the muramyl entity had a free reducing end, and a free hydroxyl on C-6), and an anhydro analogue lacking the terminal alanine. The Mr values were 939 and 850, respectively. Methyl esters were prepared, and these were also acetylated. The mass spectra of the methyl ester of Mr = 921 displayed an increase in Mr of 42 (i.e. 3 X 14), indicating the presence, originally, of three free carboxyls. Acetylation increased Mr by a further 168 units (i.e. 4 X 42), indicating 4 hydroxyl or amino groups. These data are consistent with the structure cited above for the main entity of FSu. Similar confirmatory results were obtained for the two minor constituents described above. These operations were worked out on natural muramyl peptides of known structure, obtained from other sources, and the data are given for comparison.

Peptidoglycans as promoters of slow-wave sleep. II. Somnogenic and pyrogenic activities of some naturally occurring muramyl peptides; correlations with mass spectrometric structure determination.

The structures of components of the sleep-promoting material purified from human urine were established by fast atom bombardment-mass spectrometry, as reported in the accompanying paper (Martin, S. A., Karnovsky, M. L., Krueger, J. M., Pappenheimer, J. R., and Biemann, K. (1984) J. Biol. Chem. 259, 12652-12658). We report here that two substances isolated from that preparation, viz. N-acetylglucosaminyl-1,6 -anhydro-N-acetylmuramyl-Ala-gamma-Glu-diaminopimelyl-Ala) and that compound lacking the terminal alanine, are active as somnogens. Cerebro-intraventricular administration of 1 pmol of the glycotetrapeptide was sufficient to induce prolonged excess sleep in rabbits. A similar substance obtained from Brevibacterium divaricatum in which the free carboxyls of the glutamic and diaminopimelic moieties, indicated above, were amidated (N-acetylglucosaminyl-1,6-anhydro-N-anhydromura-myl-Ala-iso- Gln- epsilon-amido-diaminopimelyl -Ala-Ala) was not active as a promoter of slow-wave sleep. Deamidation of this peptide to a mixture of the free dicarboxylic forms produced a somnogenic substance. Our findings show that in addition to the muramyl form of peptidoglycan monomers, the anhydro muramyl form, with no reducing end, is compatible with somnogenic activity. Furthermore, the data obtained with a natural product amplify our earlier observations with smaller synthetic molecules of the importance of amidation/deamidation in the structure-activity relationships of muramyl peptides.

Site of action of sleep-inducing muramyl peptide isolated from human urine: microinjection studies in rabbit brains.

1. Sleep-promoting muramyl peptide purified from human urine (SPU) was injected through chronically implanted guide tubes into defined regions of the basal forebrain and brain stem of unanesthetized rabbits. Electroencephalogram (EEG) and body movements were recorded subsequent to each injection. 2. Of 52 injection sites, there were 8 in which microinjection of SPU induced significant increases of slow-wave sleep (SWS) for 5 or more hours. Each active site was tested twice with SPU and once with vehicle control. 3. Seven of the active sites were located in a region extending from the basal forebrain at the level of the optic chiasm to the mesodiencephalic junction. No active sites were found in the lower brain stem. 4. The behavioral and EEG characteristics of the excess SWS induced by microinjection of SPU appeared normal and similar to the excess sleep that follows intraventricular infusions of sleep factor. However, the responses occurred sooner after injections into an active site than after infusion into a lateral ventricle.