Confocal microscopical analysis of epithelial cell heterogeneity in mouse Peyer's patches

Summary Cyanine dye fluorescence and alkaline phosphatase activities have been compared directly by confocal microscopy in a wide variety of cells present in the follice-associated epithelium of the mouse Peyer's patch to test the hypothesis that antigen-transporting M cells have a low membrane potential. In order to make these comparisons it was first necessary to equilibrate living tissue with the membrane potential sensitive dye DIOC₅(3), fix with glutaraldehyde and then incubate the fixed tissue with naphthol AS-BI phosphate, a substrate which is hydrolysed by alkaline phosphatase present in the luminal membrane of these epithelial cells. Naphthol AS-BI produced by this reaction, is then coupled to Fast Red TR diazonium salt at the site of hydrolysis. Selecting the 488 nm wavelength of the argon laser source then allows one to measure alkaline phosphatase activities as Fast Red absorbance and membrane potentials by DIOC₅(3) fluorescence.Results obtained show a linear correlation between membrane potential and alkaline phosphatase activity. Relative lack of alkaline phosphatase activity, determined in fixed tissue, has been used previously to identify antigen-transporting M cells (Smithet al., 1987). The present work shows that it is now possible to recognize these cells in living tissue by measurement of DIOC₅(3) fluorescence. The possible importance of this finding in providing a way to study cell surface-antigen interactions taking place in living tissue is discussed.     

Electrical parameters in gallbladders of different species their contribution to the origin of the transmural potential difference

Summary Amphotericin B enhances Na⁺ conductance of the mucosal membrane of gallbladder epithelial cells and in such a way it modifies the brush border electromotive force. On this basis a method to measure cell and shunt resistances by comparing changes of the mucosal membrane potential (V _m ) and of the transmural p.d. (V _ms ) is developed. This method is applied in gallbladders of different vertebrate species (i.e. rabbit, guinea pig, goose, tortoise, toad, trout). The two tested mammals, rabbit and guinea pig, exhibited a lower shunting percentage (89–93%) than the nonmammals (96–97%), but this fact did not bring about a homogeneous positiveV _ms . This means that shunting percent contributes, but it is not the only source of differences inV _ms , in accordance with that reported by Gelarden and Rose (J. Membrane Biol. 19:37, 1974). Moreover, mammals exhibited a lower luminal resistance and a lower ratio between luminal and basolateral resistance than nonmammals. Possible causes of these differences are discussed.     

Membrane potentials of differentiating enterocytes

A positional analysis of enterocyte membrane potential has been carried out using in vitro preparations of rabbit distal ileum. Young enterocytes were found to possess a microvillar membrane potential significantly less than that seen in older enterocytes. The length of enterocyte microvilli was also found to be significantly less in younger enterocytes. It is suggested that developmental changes in membrane potential, occurring during the early stages of enterocyte differentiation, probably reflect a changed permeability to ions associated with the establishment of a fully developed microvillar membrane. Other explanations for the observed findings are also considered.     

Colchicine effect on the permeability of the whole epithelium and of isolated cells of frog skin

The effect of 2×10^–5 M colchicine on epithelial cells isolated from frog skin was investigated. Three hours of treatment with colchicine did not change either Na⁺ and K⁺ content of isolated cells or nonelectrolyte permeability. When ADH (50 mU/ml) was added, thiourea uptake values became greater than without the hormone; the same values were found in the cells previously treated with colchicine. Na⁺ transepithelial transport, measured by means of short-circuit current, was inhibited by the antimitotic agent both under control conditions and after ADH stimulation. These results support the view that colchicine does not directly affect ADH action on membrane permeability, but influences some mechanism that controls ADH action on transepithelial transport. Intercellular junctions appear to be the location of such a mechanism.     

Zinc status and immune system relationship

The essentiality of zinc for humans was first documented by Prasad in the 1960s. The main clinical manifestations associated with zinc deficiency are growth retardation, hypogonadism, diarrhea, and increased susceptibility to infectious diseases. Thus, in the past 25 yr, there was an increased interest of researchers in studying the role of zinc in human immunity. Although mechanistic research has been carried out using animal models, there are several studies in humans with similar results. This work is an attempt to review the information available in this field to understand the important role that zinc plays in the normal development and function of the immune system.     

Extracellular space determination in gallbladder mucosa

Sucrose seems to be the best marker of the extracellular space in gallbladder mucosa. Inulin space is always smaller than sucrose space and the difference seems to be due to a sweeping-away effect in the intercellular channels as metabolic inhibitors and ouabain reduce it. Mannitol space continuously increases and this substance is likely to enter the cells.     

Positional dependence of enterocyte membrane potential in hamster and rabbit enterocytes

A technique is described allowing microelectrode impalement of enterocytes located at known positions along intestinal villi from rabbits and hamsters. Using this technique a 5 mV hyperpolarization in membrane potential is shown to occur as enterocytes migrate over the basal third of intestinal villi. The villus structure of the hamster ileum is similar to the rabbit, but the enterocyte lifespan in these two tissues differs considerably (enterocyte migration rates of 17.6 and 6.3 microns hr-1 for hamster and rabbit respectively). A correlation was found between the position an enterocyte occupied on the crypt-villus axis and the developmental state of the membrane potential. No such correlation existed when making comparisons on a time basis. These results are discussed both in terms of what is now known concerning different aspects of enterocyte development and in relation to what type of control mechanism might be generally responsible for initiating differentiation in this tissue.     

Allogenic stimulus induces prostaglandin and thromboxane production in T lymphocytes

We have examined the influence of an allogeneic stimulus on T lymphocyte prostanoid synthesis. PGE2 and TXB2 (the stable product of TXA2) were determined by radioimmunoassay. When T cells were derived from alloimmunized animals, the production of PGE2 and TXA2 was significantly higher than that of non-immunized cells. Moreover, T immune lymphocytes in the presence of the immunized alloantigen showed an increment in prostanoid production. We propose that the allogeneic stimulus provides a signal to the T lymphocytes for an increase in prostanoid synthesis.     

Further analysis of transcytosis of free polypeptides and polypeptide-coated nanobeads in rabbit nasal mucosa.

In rabbit nasal mucosa, free polypeptides and polypeptide-coated nanospheres are actively absorbed by the M cells present in specialized areas of the epithelium. Because polypeptide-coated nanosphere transport was abolished in the presence of free polypeptides, free polypeptides and polypeptide-coated nanospheres are shown here to compete. Fluxes of polypeptide-coated nanospheres with 356, 490, and 548 nm diameters have been compared. BSA-coated beads were poorly transported, at the same rate, when bead diameters were 356 or 490 nm [net flux of approximately 2-2.5 x 10(6) nanospheres (nan). cm(-2) x h(-1)]; however, their net transport largely increased toward a value of 25 x 10(6) nan. cm(-2) x h(-1) at a diameter of 548 nm. Insulin-coated beads displayed a net flux that was significantly higher than BSA-coated beads but equally were transported at the same rate (net flux of approximately 8.0 x 10(6) nan. cm(-2) x h(-1)) at diameters of 356 or 490 nm; once again, their net flux significantly increased toward a value of 25 x 10(6) nan. cm(-2) x h(-1), if the bead diameter was 548 nm. Insulin plus anti-insulin IgG-coated 490-nm-diameter beads displayed a very high net flux, although not yet saturating (approximately 60 x 10(6) nan. cm(-2) x h(-1)); however, a significantly lower saturated net flux (once again approximately 25 x 10(6) nan. cm(-2) x h(-1)) was shown with 548-nm-diameter beads. In conclusion, 1) in the range of 356-490 nm diameter, net transport was independent of bead diameter and, conversely, largely dependent on the coating polypeptides, and 2) at 548 nm diameter, nanospheres tended to be transferred at similar rates independently of coating kind and the maximal net transport capacity of the mucosa was reduced. The suspension viscosity largely increased with 548-nm polypeptide-coated nanospheres; this fact is hypothetically proposed to be the cause of these events.