Freeze-fracture study of the junctional complexes of human and rabbit thyroid follicles

Summary Zonulae occludentes, gap junctions and desmosomes have been demonstrated in replicas of freeze-fractured follicular cells of normal human and rabbit thyroid glands. The zonulae occludentes between the human follicular cells are composed of two to eight strands, which completely separate the intercellular space from the follicular lumen. Four to twelve or more strands are visible between the follicular cells of the rabbit thyroid gland.In the meshes of the zonulae occludentes as well as below them, gap junctions are present. They are numerous on the fracture faces of the human follicular cell membranes, but infrequent in those of the rabbit.Aggregates of particles related to desmosomes are found in the deeper meshes of the zonulae occludentes or close to them.     

Separation of synaptic junctional complexes from thin layers of rabbit cerebral cortex

Synaptic junctional fractions were separated from rabbit brain by procedures based on combining the methods of Cotman and Taylor [4], Orosz et al. [16, 17] and Lisman et al. [13]. Thin layers of cerebral cortices were homogenized to obtain a crude mitochondrial-synaptosomal fraction. The sedimentation rates of mitochondria and mitochondria containing synaptosomes were increased by raising the density of mitochondria with an insoluble dense formazan deposit inside mitochondria after iodo-nitrotetrazolium treatment. The synaptic plasma membrane fraction isolated by this method contained no mitochondrial contamination. After Triton X-100 treatment the insoluble residues of the detergent were centrifuged through discontinuous sucrose gradients. A great enrichment of morphologically identifiable intact synaptic junctions was observed in some of the obtained interface layers.     

Factors in the movement of hexose across the intestine of Ascaris suum

Trichloroacetic acid extractable carbohydrate of the intestine of Ascaris suum decreases rapidly when ribbons of the tissue are incubated in a basal salt solution. After 10 min incubation endogenous carbohydrate is 32% and after 80 min it is 19% of the "zero" time control value. In contrast, there is approximately a 2-fold increase in the endogenous carbohydrate of tissue that is incubated in saline with glucose. The increase occurs within the first 5 min and is maintained throughout an 80-min incubation period. Sac preparations of the intestine that are preincubated with glucose and incubated without glucose move 3-0-methylglucose from the luminal to the pseudocoelomic fluid at a rate that is comparable initially to the rate of movement measured for sac preparations that are incubated in saline with glucose. After 10 min the rate decreases. The addition of glycogen or trehalose to the saline bathing the pseudocoelomic side of sac preparations does not facilitated the movement of 3-0-methylglucose. Collectively, the results support the suggestion that the limited movement of 3-0-methylglucose across intestinal sac preparations that are incubated without glucose is due to the tissue's limited carbohydrate reserve and its rapid depletion in vitro.     

Studies on perineural junctional complexes and the sites of uptake of microperoxidase and lanthanum in the cockroach central nervous system

The perineurial junctional complexes in the nerve cord of Periplaneta americana have been shown to consist of septate desmosomes, extensive gap junctions and relatively limited regions of tight junctions. Microperoxidase (M.W. 1,900) undergoes limited intercellular penetration into the septate desmosomes. Lanthanum penetrates both the septate desmosomes and gap junctions. It is concluded that the restricted access of these substances to the underlying extracellular spaces results from the presence of the perineurial tight junctions. These results contrast with those for small peripheral nerves, which lack equivalent junctional complexes, and in which the extracellular spaces are found to be accessible to externally applied lanthanum. The results are discussed in relation to current concepts of the insect blood-brain barrier.     

The formation of septate-like junctional complexes between lymphoid cells in vitro.

During the initial 48 hr of incubation in immunized Mishell-Dutton spleen cell cultures, most lymphocytes exist as single cells or an occasional pair of cells (doublets). Ultrastructural examination of the area between those cells forming doublets revealed a septate-like junctional complex occurring over large portions of the plasmalemma. This junctional zone persisted in areas where extensive cytoplasmic interdigitation among cell processes was evident. Approximately 1% of the lymphocytes isolated from the top fraction of a discontinuous fetal calf serum gradient were involved in formation of doublets. The majority of these doublets showed evidence of junctional interaction. Moreover, this septate-like zone of adhesion was seen using several different fixatives and stains and remained intact after osmotic cell disruption. The junction was not demonstrable by using freeze-fracture techniques and therefore must be limited to interactions within the surface of the cells.     

The movement of ion pairs across bilayer lipid membranes.

If a polyhalide concentration gradient exists across a bilayer lipid membrane (BLM), ion pair movement occurs. The term ion pair indicates a lipid soluble complex of cation and anion with stoichiometry dictated by the respective charges. In a mixture of metal halide (MX_n, X = I, Cl, Br) and iodine, the ion pair is of the form M(I₂X)_n. The flux of ion pairs was monitored by measuring the flow of metal ions or polyhalide ions across the BLM. The flux of ion pairs across the BLM depended on cation crystal radius, fluidity of the membrane, strength of the ion pair complex and on the osmotic gradient (i.e., there exists a coupling between water and ion pair fluxes). The relationship between ion pairing and the electrical conductivity of BLM is briefly discussed.     

Pseudo-streaming potentials in Necturus gallbladder epithelium. II. The mechanism is a junctional diffusion potential

The mechanisms of apparent streaming potentials elicited across Necturus gallbladder epithelium by addition or removal of sucrose from the apical bathing solution were studied by assessing the time courses of: (a) the change in transepithelial voltage (Vms). (b) the change in osmolality at the cell surface (estimated with a tetrabutylammonium [TBA+]-selective microelectrode, using TBA+ as a tracer for sucrose), and (c) the change in cell impermeant solute concentration ([TMA+]i, measured with an intracellular double-barrel TMA(+)-selective microelectrode after loading the cells with TMA+ by transient permeabilization with nystatin). For both sucrose addition and removal, the time courses of Vms were the same as the time courses of the voltage signals produced by [TMA+]i, while the time courses of the voltage signals produced by [TBA+]o were much faster. These results suggest that the apparent streaming potentials are caused by changes of [NaCl] in the lateral intercellular spaces, whose time course reflects the changes in cell water volume (and osmolality) elicited by the alterations in apical solution osmolality. Changes in cell osmolality are slow relative to those of the apical solution osmolality, whereas lateral space osmolality follows cell osmolality rapidly, due to the large surface area of lateral membranes and the small volume of the spaces. Analysis of a simple mathematical model of the epithelium yields an apical membrane Lp in good agreement with previous measurements and suggests that elevations of the apical solution osmolality elicit rapid reductions in junctional ionic selectivity, also in good agreement with experimental determinations. Elevations in apical solution [NaCl] cause biphasic transepithelial voltage changes: a rapid negative Vms change of similar time course to that of a Na+/TBA+ bi-ionic potential and a slow positive Vms change of similar time course to that of the sucrose-induced apparent streaming potential. We conclude that the Vms changes elicited by addition of impermeant solute to the apical bathing solution are pseudo-streaming potentials, i.e., junctional diffusion potentials caused by salt concentration changes in the lateral intercellular spaces secondary to osmotic water flow from the cells to the apical bathing solution and from the lateral intercellular spaces to the cells. Our results do not support the notion of junctional solute-solvent coupling during transepithelial osmotic water flow.     

The movement of amino acids into rabbit oviductal fluid.

Twenty-three amino acids appeared linearly in oviduct fluid to reach an average concentration 27% of that in the plasma, with glycine and alanine present in greatest amount. In contrast, the concentration of urea in the fluid approached its plasma concentration after 1 h, and remained at this level for the duration of the experiment. The oviduct was unable to bring the overall uphill transport of amino acids from plasma to oviduct lumen. The amounts of amino acids appearing in the oviduct lumen of rabbits injected 3 days previously with hCG were mostly similar to those of the controls, but the movements of 6 neutral amino acids appeared to be enhanced relative to their plasma concentrations following ovulation.     

A calcium-sensitive lanthanum inhibition of amoeboid movement

The effects of lanthanum ions upon amoeboid movement and upon the redistribution of dense cytoplasmic inclusions following centrifugation were investigated in Amoeba discoides. Treatment with low lanthanum concentrations produced a characteristic inhibited condition in which locomotion ceased while saltatory movements continued. The resorting of stratified cytoplasm by local internal cytoplasmic flow was unaffected. This contrasted with p-hydroxymercuribenzoate treatment which inhibited both locomotion and saltatory movements. The inhibitory effects of lanthanum ions were antagonised by calcium ions but not by magnesium ions. It is reasoned that lanthanum competes for a peripheral binding site thereby disrupting a calcium-mediated regulatory mechanism of amoeboid movement.     

Osmotic Flow of Water across Permeable Cellulose Membranes

Direct measurements have been made of the net volume flow through cellulose membranes, due to a difference in concentration of solute across the membrane. The aqueous solutions used included solutes ranging in size from deuterated water to bovine serum albumin. For the semipermeable membrane (impermeable to the solute) the volume flow produced by the osmotic gradient is equal to the flow produced by the hydrostatic pressure RT ΔC, as given by the van't Hoff relationship. In the case in which the membrane is permeable to the solute, the net volume flow is reduced, as predicted by the theory of Staverman, based on the thermodynamics of the steady state. A means of establishing the amount of this reduction is given, depending on the size of the solute molecule and the effective pore radius of the membrane. With the help of these results, a hypothetical biological membrane moving water by osmotic and hydrostatic pressure gradients is discussed.     

The mechanism of cation permeation in rabbit gallbladder

Summary The questions underlying ion permeation mechanisms, the types of experiments available to answer these questions, and the properties of some likely permeation models are examined, as background to experiments designed to characterize the mechanism of alkali cation permeation across rabbit gallbladder epithelium. Conductance is found to increase linearly with bathing-solution salt concentrations up to at least 400mm. In symmetrical solutions of single alkali chloride salts, the conductance sequence is K⁺>Rb⁺>Na⁺>Cs⁺∼Li⁺. The current-voltage relation is linear in symmetrical solutions and in the presence of a single-salt concentration gradient up to at least 800 mV. The anion/cation permeability ratio shows little change with concentration up to at least 300mm. Ca⁺⁺ reduces alkali chloride single-salt dilution potentials, the magnitude of the effect being interpreted as an inverse measure of cation equilibrium constants. The equilibrium-constant sequence deduced on this basis is K⁺>Rb⁺>Na⁺∼Cs⁺∼Li⁺. These results suggest (1) that the mechanism of cation permeation in the gallbladder is not the same as that in a macroscopic ion-exchange membrane; (2) that cation mobility ratios are closer to one than are equilibrium-constant ratios; (3) that the rate-limiting step for cation permeation is in the membrane interior rather than at the membrane-solution interface; and (4) that the rate-controlling membrane is one which is sufficiently thick that it obeys microscopic electroneutrality.     

Water movement across split frog skin.

A net inward fluid reabsorption (salt-linked flow) has been observed in isolated skin epithelium (split skin) with the same magnitude as in whole skin when identical NaCl Ringer solutions were used to bathe both sides. Split skins also respond to a hyperosmotic sucrose solution bathing the outer (epithelial) surface by generating an outward osmotic flow. A non-linear relationship between osmotic flow and the osmotic gradient has been found in split skin similar to that found in whole skin.