Sodium and Sugar Fluxes across the Mucosal Border of Rabbit Ileum

Unidirectional influxes of sugars and Na from muscosal solution into the cells of rabbit ileum have been examined. The influxes of glucose, galactose, and 3-0-methyl glucose (3 MG) follow Michaelis-Menten type kinetics and are markedly dependent on the presence ofNa in the mucosal solution. For 3 MG, reduction of Na concentration causes a decrease in maximal rate of influx and little change in the "apparent Michaelis constant." There appeared to be little mediated entry of 3 MG into the cells from Na-free solution. The influx of Na was increased by the presence of 3 MG in the mucosal solution and at all Na concentrations tested, there was a 1:1 ratio between sugar influx and the sugar-dependent Na influx. On the basis of these observations, a model has been developed for the sugar transport system involving a transport site that combines with both sugar and Na.     

Three Odorant-binding Proteins from Rabbit Nasal Mucosa

Following the purification of an odorant-binding protein (OBP)from rabbit nasal mucosa, we have identified, purified and partiallycharacterized two additional OBPs from the nasal tissue of thesame animal species. OBP-II is a monomer of 21 kDa and isoelectricpoint 4.2; OBP-III is a dimer with subunits of 23 kDa and isoelectricpoint 4.8. Like OBP-I, both these new members bind the odorant2-isobutyl-3-methoxypyrazine. The partial amino acid sequencesof the three OBPs, determined by Edman degradation, confirmthat they are members of the OBP family, but reveal poor similaritybetween them. However, higher similarity is found between eachOBP and other members of the lipocalin family. In particular,OBP-I is most similar to bovine OBP (55% identity in the N-terminalregion), OBP-II is >50% identical, limited to its first 18amino acids, to mouse OBP-I and porcupine OBP-II, while OBP-IIIshares 26 out of the first 40 amino acids with major urinaryprotein (MUP) 4, a member of the mouse salivary proteins. Thepossible role of these proteins in olfactory transduction isalso discussed. Chem. Senses 22: 383–390, 1997.     

Characteristics of the Amino Acid Transport System in the Mucosal Border of Rabbit Ileum

The specificity of the neutral amino acid transport system in the brush border was examined by studying the ability of amino acid analogues to inhibit the unidirectional influx of phenylalanine from mucosal solution into the cells. Effects were evaluated in terms of the affinity of various substrates for the amino acid site in the transport system. The affinity of amino acids for the site was proportional to the number of carbon atoms in the side chain. Electron-withdrawing substituents in the ring of phenylalanine increased affinity and electron-releasing groups decreased affinity. Removal of the α-amino group from phenylalanine decreased affinity by a factor of approximately 50 and removal of the carboxyl group decreased affinity 12-fold. Effects on affinity of variations in the side chain of the amino acid can be comparable in magnitude to that of the carboxyl group. The effect of sodium ion on the transport system appears to be similar for all compounds tested.     

Effect of Inhibitors on Alanine Transport in Isolated Rabbit Ileum

The effects of metabolic inhibitors and ouabain on alanine transport across rabbit ileum, in vitro, have been investigated. Net transport of alanine and Na across short-circuited segments of ileum is virtually abolished by cyanide, 2,4-dinitrophenol, iodoacetate, and ouabain. However, these inhibitors do not markedly depress alanine influx from the mucosal solution, across the brush border, into the intestinal epithelium, and they do not significantly affect the Na dependence of this entry process. The results of this investigation indicate that: (a) the Na dependence of alanine influx does not reflect a mechanism in which the sole function of Na is to link metabolic energy directly to the influx process; and (b) the inhibition of net alanine transport across intestine is, in part, the result of an increased rate coefficient for alanine efflux out of the cell across the brush border. Although these findings do not exclude a direct link between metabolic energy and alanine efflux, the increased efflux may be the result of the increased intracellular Na concentration in the presence of these inhibitors. The results of these studies are qualitatively consistent with a model for alanine transport across the brush border which does not include a direct link to metabolic energy.     

Sodium Transport in Triads Isolated from Rabbit Skeletal Muscle

Triads and transverse tubules isolated from mammalian skeletal muscle actively accumulated Na+ in the presence of K+ and Mg-ATP. Active Na+ transport exhibited a fast single-exponential phase, lasting 2 min, followed by slower linear uptake that continued for 10 minutes. Valinomycin stimulated Na+ uptake, suggesting it decreased a pump-generated membrane potential gradient (Vm) that prevented further Na+ accumulation. At the end of the fast uptake phase transverse tubule vesicles incubated in 30 mM external [Na+] attained a ratio [Na+]in/[Na+]out=13.4. From this ratio and the transverse tubule volume of 0.35 microl/mg protein measured in this work, [Na+]in=400 mM was calculated. Determinations of active K+ transport in triads, using 86Rb+ as tracer, showed a 30% decrease in vesicular 86Rb+ content two minutes after initiating the reaction, followed by a slower uptake phase during which vesicles regained their initial 86Rb+ content after 10 minutes. Transverse tubule volume increase during active Na+ transport-as shown by light scattering changes of isolated vesicles--presumably accounted for the secondary Na+ and 86Rb+ uptake phases. These combined results indicate that isolated triads have highly sealed transverse tubules that can be polarized effectively by the Na+ pump through the generation of significant Na+ gradients.     

The Sodium-Alanine Interaction in Rabbit Ileum : Effect of sodium on alanine fluxes

The model of the interaction between Na and alanine at the mucosal border of rabbit ileum has been tested further by examining the efflux of alanine from the cells toward the mucosal solution. Alanine efflux shows a tendency toward saturation as cellular alanine concentration increases and is influenced by cellular Na concentration. A decrease in cell Na concentration causes an increase in the apparent Michaelis constant with little change in maximal efflux rate. Studies on strips of mucosa treated with ouabain or cyanide showed that the direction of net alanine transfer between the cells and the medium is determined by the direction of the Na concentration difference. The cells extrude alanine against a concentration difference when cell [Na] exceeds medium [Na] and accumulate alanine when cell [Na] is less than medium [Na]. The observations are consistent with the model previously suggested involving a transport site that combines with and translocates both Na and alanine, and with the concept that the Na concentration difference between mucosal solution and cytoplasm provides at least part of the energy for active transport of alanine.     

Ion Transport in Isolated Rabbit Ileum : I. Short-circuit current and Na fluxes

The transmural potential difference, short-circuit current, and Na fluxes have been investigated in an in vitro preparation of isolated rabbit ileum. When the tissue is perfused with a physiological buffer, the serosal surface is electrically positive with respect to the mucosal surface and the initial potential difference in the presence of glucose averages 9 mv. Unidirectional and net Na fluxes have been determined under a variety of conditions, and in each instance, most if not all of the simultaneously measured short-circuit current could be attributed to the active transport of Na from mucosa to serosa. Active Na transport is dependent upon the presence of intact aerobic metabolic pathways and is inhibited by low concentrations of ouabain in the serosal medium. A method is described for determining whether a unidirectional ionic flux is the result of passive diffusion alone, in the presence of active transport of that ion in the opposite direction. Using this method we have demonstrated that the serosa-to-mucosa flux of Na may be attributed to passive diffusion with no evidence for the presence of carrier-mediated exchange diffusion or the influence of solvent-drag.     

Sodium Flux in Necturus Proximal Tubule under Voltage Clamp

Na transport and electrical properties of Necturus renal proximal tubules were analyzed, in vivo, by a voltage clamp method which utilizes an axial electrode in the tubule lumen for passage of current and simultaneous determination of net fluid (or Na) flux by the split droplet method. When the average spontaneous transepithelial potential difference of –8 mv (lumen negative) was reduced to zero by current passage, net Na flux doubled from a mean of 107 to 227 pmoles/cm² per sec. The relationship between flux and potential over the range –25 to +10 mv was nonlinear, with flux equilibrium at –15 mv and droplet expansion at more negative values. Calculated Na permeability at flux equilibrium was 7.0 x 10^–6 cm/sec. Voltage transients, similar to those caused by intraepithelial unstirred layers, were observed at the end of clamping periods. Tubular electrical resistance measured by brief square or triangle wave pulses (<100 msec) averaged 43 ohm cm². The epithelial current-voltage relationship was linear over the range –100 to +100 mv, but displayed marked hysteresis during low frequency (<0.04 Hz) triangle wave clamps. The low transepithelial resistance and large opposing unidirectional ion fluxes suggest that passive ionic movements occur across extracellular shunt pathways, while the voltage transients and current-voltage hysteresis are consistent with the development of a local osmotic gradient within epithelium.     

Components of Sodium and Chloride Flux Across Toad Bladder

The effect of transepithelial potential difference (ψ) on Na and Cl flux across toad bladder was assessed by measuring isotopic flux between identical media at various values of ψ. The contribution of edge damage to ionic permeability was eliminated, resulting in relatively high spontaneous ψ (-97 ±4 mv) and low electrical conductance g. Bidirectional Na fluxes were measured simultaneously. Unidirectional Cl fluxes were measured in paired hemibladders at ψ = 0 mv or -97 mv. Net Na flux J_Na, at ψ = 0 mv, was slightly less than short-circuit current (SCC). At ψ = -97 mv, J_Na averaged 17% of SCC, and was sometimes zero. ΔJ_Na/Δψ (= g₊) averaged 60% of g between -97 mv and +75 mv; at -150 mv, g₊ fell, indicating rectification. Analysis of unidirectional Na fluxes indicates low passive conductance (1.5 μmho/mg wet weight), a bidirectional, electrically neutral flux of approximately 0.13 μa/mg, and relatively large conductance of the active transport path at ψ ≥ -97 mv. The absence of appreciable transstimulation of serosal (S)-to-mucosal (M) Na flux (in response to increasing mucosal Na concentration) indicates that the electrically neutral flux is not exchange diffusion in the usual sense. Analysis of Cl fluxes indicates similar values for passive conductance and neutral flux, suggesting linked neutral flux of Na and Cl. Either the electromotive force of the Na pump E, its conductance g_a, or both are strong functions of ψ. The product of these two quantities, Eg_a, is a measure of the “transport capacity” at any given value of ψ, independent of the direct effect of ψ on J_Na through the pump path. Eg_a varies with ψ. Hence estimation of the net Na flux or current at any one value of ψ, including ψ = 0, fails to reveal the maximal transport capacity of the pump, its resting electromotive force (when J_Na = 0 through the pump), or the dependence of transport capacity on potential.     

Pre-Analytical Determination of the Effect of Extended Warm or Cold Ischaemia on RNA Stability in the Human Ileum Mucosa

The use of banked human tissue, obtained with informed consent after elective surgical procedures, represents a powerful model for understanding underlying mechanisms of diseases or therapeutic interventions and for establishing prognostic markers. However, donated tissues typically have varying times of warm ischaemia in situ due to blood arrest or cold ischaemia due to procurement and transportation. Hence, before using these tissues, it is important to carry out pre-analytical studies to ensure that they are representative of the in vivo state. In particular, tissues of the gastrointestinal tract have been thought to have low RNA stability. Therefore, this study aimed to determine if extended warm or cold ischaemia times and snap-freezing or banking in RNA stabilization solution affects RNA integrity or gene expression in human ileum mucosa. In short, ileum mucosa was collected for up to 1.5 h and 6 h of simulated warm or cold ischaemia respectively. Subsequently, RNA integrity and gene expressions were determined. It was found that RNA integrity remained high over the course of warm and cold ischaemia examined and there were in general no significant differences between snap-freezing and banking in RNA stabilization solution. Following the same trend, there were in general no significant changes in gene expressions measured (MYC, HIF1α, CDX, HMOX1 and IL1β). In conclusion, RNA in the ileum mucosa is maintained at a high integrity and has stable gene expression over the examined time course of warm or cold ischaemia when banked in RNA stabilization solution or snap-frozen in liquid nitrogen. As the average warm and cold ischaemia times imposed by surgery and the process of tissue banking are shorter than the time period examined in this study, human ileum mucosa samples collected after surgeries could be used for gene expression studies.     

Cellular Inhomogeneity in Dog Red Cells As Revealed by Sodium Flux

Unidirectional ²⁴Na fluxes across the dog red blood cell membrane were measured. The kinetics were incompatible with a single time constant but could be accounted for in terms of a two-series compartment cell model, with approximately 1% of cell Na in the outer compartment. Dog red blood cells are known to be inhomogeneous in their Na and K permeabilities. Theoretical analysis showed that such cellular inhomogeneity in the Na permeability coefficient might in principle account for the flux data. In order to evaluate the inhomogeneity effect, a technique based on the differential response of cells suspended in isosmolar high K buffers was devised to measure the variations in Na permeability in the cell population. A variation in the Na permeability coefficient of approximately 30% was found. This inhomogeneity is insufficient to account for the flux data.     

Ion Transport in Isolated Rabbit Ileum : II. The interaction between active sodium and active sugar transport

The addition of actively transported sugars to the solution bathing the mucosal surface of an in vitro preparation of distal rabbit ileum results in a rapid increase in the transmural potential difference, the short-circuit current, and the rate of active Na transport from mucosa to serosa. These effects are dependent upon the active transport of the sugar per se and are independent of the metabolic fate of the transported sugar. Furthermore, they are inhibited both by low concentrations of phlorizin in the mucosal solution and by low concentrations of ouabain in the serosal solution. The increase in the short-circuit current, ΔI_sc, requires the presence of Na in the perfusion medium and its magnitude is a linear function of the Na concentration. On the other hand, ΔI_sc is a saturable function of the mucosal sugar concentration which is consistent with Michaelis-Menten kinetics suggesting that the increase in active Na transport is stoichiometrically related to the rate of active sugar transport. An interpretation of these findings in terms of a hypothetical model for intestinal Na and sugar transport is presented.     

Isolation of Neuropeptide-Containing Vesicles from the Guinea Pig Ileum

Three distinct vesicle fractions enriched 40-60 times in the neuropeptides substance P, somatostatin, and vasoactive intestinal peptide (VIP) were prepared from the myenteric plexus of guinea pig ileum by density gradient centrifugation in a small zonal rotor. Mean densities (in g X ml-1) and diameters (in nm) of the three classes of vesicles were: substance P, 1.123, 65; somatostatin, 1.138, 37; VIP, 1.148, 110; standard deviations were about 5%. These peaks were distinct from the peak of acetylcholine-containing vesicles of density 1.066 g X ml-1 and diameter 61 nm. When a relatively mild method of homogenization was used a second peak of acetylcholine appeared in the same region of the gradient as VIP and the VIP was larger. This may represent a class of vesicles containing both acetylcholine and VIP, though cosedimentation of two classes of vesicles of almost the same density and similar fragility, one containing VIP and the other acetylcholine, cannot be entirely excluded on present evidence.     

Ionic Conductances of Extracellular Shunt Pathway in Rabbit Ileum : Influence of shunt on transmural sodium transport and electrical potential differences

The unidirectional influxes of Na, K, and Cl into isolated strips of rabbit ileum are comprised of movements across the mucosal membrane of the epithelial cells and ionic diffusion into an extracellular shunt pathway. A large fraction of the Na influx across the mucosal membrane alone is inhibited by Li, suggesting the participation of a carrier mechanism in the influx process. The partial ionic shunt conductances of Na, K, and Cl account for at least 82% of the total tissue conductance. The calculated shunt permeabilities (P) are (in centimeters per hour) P_K = 0.040, P_Na = 0.035, and P_Cl = 0.019, so that P_K:P_Na:P_Cl = 1.14:1.00:0.55. Diffusion potentials across the tissue resulting from isotonic replacement of NaCl in the mucosal solution with mannitol or KCl are described by the Goldman constant-field equation together with the above permeabilities of the shunt pathway. These observations are not consistent with permeation through a fixed-charge pore but can be explained by a model featuring constant ionic partition into a neutral-polar pore that traverses the tight junction. Such a pore may be lined with either fixed dipoles or fixed dipolar ions oriented such that electronegative groups influence the permselective properties of the diffusion pathway. The essential feature of both models is that electroneutrality is maintained by means of fixed membrane components and does not depend upon the presence of mobile counterions.     

Binding Characteristics of the Muscarinic Receptor Subtype in Rabbit Pancreas

Abstract

The muscarinic receptor in the rabbit pancreas was characterized with the use of the labeled ligand (³H)-(-)-quinuclidinyl-benzylate ((³H)-(-)-QNB). Specific binding of (³H)-(-)-QNB to pancreatic acini was found to be reversible and of high affinity, with an equilibrium dissociation constant (K_D) of 68 pmol/l and a receptor density (R_T) of 170 fmol/mg protein. Agonist binding behaviour was investigated by displacement of (³H)-(-)-QNB binding by eight agonists like arecoline, arecaïdine-propargylester (APE) and carbachol, yielding only low affinity binding sites. The inhibition of (³H)-(-)-QNB binding by the selective antagonists pirenzepine, hexahydrosiladifenidol (HHSiD) and (11–(12–((diethyl - amino)- methyl)- 1– piperidinyl) acetyl)-5, 11-dihydro-6H-pyrido (2, 3–b) (1, 4) benzodiazepin-6–one) (AF-DX 116) confirmed the M₃ nature of the rabbit pancreatic receptor.     

Unique Structural Characteristics of the Rabbit Prion Protein

Rabbits are one of the few mammalian species that appear to be resistant to transmissible spongiform encephalopathies due to the structural characteristics of the rabbit prion protein (RaPrP^C) itself. Here, we determined the solution structures of the recombinant protein RaPrP^C-(91–228) and its S173N variant and detected the backbone dynamics of their structured C-terminal domains-(121–228). In contrast to many other mammalian PrP^Cs, loop 165–172, which connects β-sheet-2 and α-helix-2, is well-defined in RaPrP^C. For the first time, order parameters S² are obtained for residues in this loop region, indicating that loop 165–172 of RaPrP^C is highly ordered. Compared with the wild-type RaPrP^C, less hydrogen bonds form in the S173N variant. The NMR dynamics analysis reveals a distinct increase in the structural flexibility of loop 165–172 and helix-3 after the S173N substitution, implying that the S173N substitution disturbs the long range interaction of loop 165–172 with helix-3, which further leads to a marked decrease in the global conformational stability. Significantly, RaPrP^C possesses a unique charge distribution, carrying a continuous area of positive charges on the surface, which is distinguished from other PrP^Cs. The S173N substitution causes visible changes of the charge distribution around the recognition sites for the hypothetical protein X. Our results suggest that the ordered loop 165–172 and its interaction with helix-3, together with the unique distribution of surface electrostatic potential, significantly contribute to the unique structural characteristics of RaPrP^C.     

Rabbit mitochondrial DNA diversity from prehistoric to modern times

The mitochondrial genetic variability in European rabbit (Oryctolagus cuniculus) populations present in Europe and North Africa from 11,000 years ago to the present day has been analyzed using ancient DNA techniques. DNA was extracted from 90 rabbit bones found in 22 archaeological sites dated between the Mesolithic and recent times. Nucleotide sequences present in a variable 233-bp domain of the cytochrome b gene were compared to those present in modern-day rabbits. The results show that the structure of ancient populations of wild rabbit exhibited remarkable stability over time until the Middle Ages. At this time, a novel type of mtDNA molecule abruptly appears into most wild populations studied from France. This mtDNA type corresponds to that currently present in the domestic breeds of rabbit examined so far. The relative rapidity by which this mtDNA type established and its absence in all sites examined before 1,700 years ago lend support to the hypothesis that between 2,000 and 1,000 years ago, man may have favored the development, into all regions of France, of animals carrying this particular mtDNA molecule. The origin of such animals has still to be found: animals previously living outside of France or within France but in very restricted areas? This event was concomitant with the documented establishment of warrens after the tenth century a.d. in Europe.     

兔骨髓间充质干细胞的体外分离培养和生物学特性观察

目的:探讨兔骨髓间充质干细胞体外分离、培养和鉴定方法,观察其生物学特性.方法:采集兔股骨及胫骨骨髓组织,采用密度梯度离心法结合贴壁培养法体外分离、培养和扩增兔骨髓间充质干细胞,倒置相差显微镜观察细胞形态,绘制原代、第1、3、8代细胞生长曲线,流式细胞术检测细胞表面标志物,成骨和成脂肪诱导培养鉴定,观察细胞生物学特性.结果:培养的BMSCs呈纺锤形、长梭形,旋涡状排列、放射性生长,增值活跃.各代细胞生长曲线呈S型,细胞增值活跃.细胞表面标志物CD44分子阳性,CD34和CD45分子阴性.经成骨和成脂肪诱导后细胞碱性磷酸酶染色和油红O染色阳性.结论:成功建立了兔BMSCs体外分离、培养的有效方法,扩增的BMSCs仍保留多向分化潜能,是理想的组织工程种子细胞.