Clarification of the intercellular space phenomenon in toad urinary bladder

Summary Vasopressin has been noted to increase the size of the intercellular spaces of toad bladder epithelium even in the absence of an osmotic gradient. The present studies demonstrate that the same phenomenon may be obtained in the presence or absence of a transepithelial gradient of glutaraldehyde indicating that the effect is not a fixation artifact. Morphologic evidence is presented demonstrating continuity between the epithelium and underlying smooth muscle. The data support the concept that not only net water flow, but also changes in smooth muscle tone can appreciably affect the epithelial geometry.     

FLUID TRANSPORT IN THE RABBIT GALLBLADDER : A Combined Physiological and Electron Microscopic Study

The fine structure of the rabbit gallbladder has been studied in specimens whose functional state was undetermined, which were fixed either in situ or directly after removal from the animal; in specimens whose rate of fluid absorption was determined, either in vivo or in vitro, immediately prior to fixation; and in specimens from bladders whose absorptive function was experimentally altered in vitro. Considerable variation was found in the width of the epithelial intercellular spaces in the bladders whose functional state was undefined. In bladders known to be transporting fluid, either in vivo or in vitro, the intercellular spaces were always distended, as were the subepithelial capillaries. This distension was greatest in bladders which had been functioning in vitro. When either Na⁺ or Cl^- was omitted from the bathing media, there was no fluid transport across the wall of the gallbladder studied in vitro. The epithelial intercellular spaces of biopsies taken from several bladders under these conditions were of approximately 200 A width except for minor distension at the crests of mucosal folds. The addition of the missing ion rapidly led to the reestablishment of fluid transport and the distension of the intercellular spaces throughout most of the epithelium of these bladders. Studies of sodium localization (by fixation with a pyroantimonate-OsO₄ mixture) showed high concentrations of this ion in the distended intercellular spaces. Histochemical studies of ATPase activity showed that this enzyme was localized along the lateral plasma membrane of the epithelial cells. The analogy is drawn between the structure of the gallbladder mucosa and a serial membrane model proposed by Curran to account for coupled solute-solvent transport across epithelia. It is concluded that the intercellular compartment fulfills the conditions for the middle compartment of the Curran model and that active transport of solute across the lateral plasma membrane into the intercellular space may be responsible for fluid absorption by the gall bladder.     

THE ULTRASTRUCTURE AND DISPOSITION OF VESICULATED NERVE PROCESSES IN SMOOTH MUSCLE

The walls of the gastrointestinal tract and urinary bladder of rats were fixed in osmium tetroxide, embedded in methacrylate, and sectioned for electron microscopy. The examination of sections of smooth muscle tissue with the electron microscope reveals the presence of bundles of unmyelinated nerve fibers within the intercellular spaces. In addition, vesiculated nerve processes, bounded on their outer surfaces by delicate plasma membranes and typically containing varying quantities of synaptic vesicles and mitochondria, make intimate contact with the surface of smooth muscle cells. These nerve processes are similar in structure and disposition to nerve endings previously described in skeletal muscle, in the central nervous system, in peripheral ganglia, in receptors, and in glands. It is concluded that the relationships existing between vesiculated nerve processes and the surface of smooth muscle cells constitute neuromuscular junctions. Profiles of protrusions of smooth muscle cells are often seen protruding into the intercellular spaces. Here they occur singly or in groups, originating from one or more cells. Because of the plane of section the protrusions may sometimes appear as individual entities between the muscle cells. In such cases care must be exercised in their identification because they have characteristics similar to sectioned nerve processes which also occur in the intercellular spaces.     

CELLULAR DIFFERENTIATION IN THE URINARY BLADDER OF A EURYHALINE MARINE FISH, GILLICHTHYS MIRABILIS, IN RESPONSE TO ENVIRONMENTAL SALINITY CHANGE

The urinary bladder of a euryhaline marine teleost, Gillichthys mirabilis , was studied by light and electron microscopy. An enlargement of the mesonephric ducts forms a sac-like structure lined by an epithelium composed of two major cell types. Tall columnar cells continuous with the duct epithelium are characterized by a large number of mitochondria and well-developed rough and smooth endoplasmic reticulum. Tubular smooth endoplasmic reticulum is more developed in the basal cytoplasm and often opens into the extracellular space. A second cell type, the low cuboidal cells, forming most of the bladder epithelium, has fewer mitochondria. Basal cells are rarely observed and mucous cells are absent.
In seawater Gillichthys , cells of both types are separated by narrow intercellular spaces. In 5% seawater fish, the columnar cells show functional activation, as evidenced by an increased number of mitochondria and more extensive tubular smooth endoplasmic reticulum. No such changes were noted in the cuboidal cells; however, the lateral intercellular spaces are dilated probably owing to hypotonicity of the urine in the hypotonic environment. A functional difference between the two cell types is strongly suggested. The columnar cells may be responsible for active sodium uptake in hypotonic seawater environments.     

High‐resolution imaging of the actin cytoskeleton and epithelial sodium channel,CFTR, and aquaporin‐9 localization in the vas deferens

Vas deferens is a conduit for sperm and fluid from the epididymis to the urethra. The duct is surrounded by a thick smooth muscle layer. To map the actin cytoskeleton of the duct and its epithelium, we reacted sections of the proximal and distal regions with fluorescent phalloidin. Confocal microscopic imaging showed that the cylinder‐shaped epithelium of the proximal region has a thick apical border of actin filaments that form microvilli. The epithelium of the distal region is covered with tall stereocilia (13–18 µm) that extend from the apical border into the lumen. In both regions, the lateral and basal cell borders showed a thin lining of actin cytoskeleton. The vas deferens epithelium contains various channels to regulate the fluid composition in the lumen. We mapped the localization of the epithelial sodium channel (ENaC), aquaporin‐9 (AQP9), and cystic fibrosis transmembrane conductance regulator (CFTR) in the rat and mouse vas deferens. ENaC and AQP9 immunofluorescence were localized on the luminal surface and stereocilia and also in the basal and smooth muscle layers. CFTR immunofluorescence appeared only on the luminal surface and in smooth muscle layers. The localization of all three channels on the apical surface of the columnar epithelial cells provides clear evidence that these channels are involved concurrently in the regulation of fluid and electrolyte balance in the lumen of the vas deferens. ENaC allows the flow of Na⁺ ions from the lumen into the cytoplasm, and the osmotic gradient generated provides the driving force for the passive flow of water through AQP channels.     

PATHS OF TRANSTUBULAR WATER FLOW IN ISOLATED RENAL COLLECTING TUBULES

The cells of perfused rabbit collecting tubules swell and the intercellular spaces widen during osmotic flow of water from lumen to bath induced by antidiuretic hormone (ADH). Ouabain had no influence on these changes. In the absence of net water flow intercellular width was unaffected when tubules were swollen in hypotonic external media. Therefore, during ADH-induced flow widening of intercellular spaces is not a consequence of osmotic swelling of a closed intercellular compartment containing trapped solutes, but rather is due to flow of solution through the channel. Direct evidence of intercellular flow was obtained. Nonperfused tubules swollen in hypotonic media were reimmersed in isotonic solution with resultant entry of water into intercellular spaces. The widened spaces gradually collapsed completely. Spaces enlarged in this manner could be emptied more rapidly by increasing the transtubular hydrostatic pressure difference. In electron micrographs a path of exit of sufficient width to accommodate the observed rate of fluid flow was seen at the base of the intercellular channel. It is concluded that the intercellular spaces communicate with the external extracellular fluid and that water, having entered the cells across the luminal plasma membrane in response in ADH, leaves the cells by osmosis across both the lateral and basilar surface membranes.     

RADIOAUTOGRAPHIC LOCALIZATION OF SODIUM PUMP SITES IN RABBIT INTESTINE

Direct demonstration of the cellular location of sodium pumping constitutes a key problem in the solution of intestinal sodium absorption. Utilizing silicone-impregnated epoxy sections of freeze-dried, osmium-fixed tissue, ouabain-³H and inulin-³H light microscope radioautographs have been produced which show that: lateral but not brush border membranes of rabbit small intestine bind ouabain-³H (high specific activity) with an affinity so great that a subsequent washing in ouabain-free medium has little effect on binding; lateral membrane binding is not apparent with low specific activity ouabain-³H, and inulin-³H and ouabain-³H (low specific activity) in the cores of the villi do not equilibrate with the intercellular spaces. Preliminary tracer measurements of ouabain-³H and inulin-¹⁴C spaces also agree with these findings As ouabain is a specific inhibitor of active sodium transport, these observations provide direct support for the view that lateral membrane pumping of sodium into the intercellular spaces causes, through osmotic forces on water, a flow of fluid out of these spaces into the interstitium.     

Ultrastructural modifications of frog urinary bladder epithelium under the influence of hypertonic media

Summary The frog urinary bladder undergoes a marked increase in its water permeability when incubated in hypertonic media. Many similarities are found between this effect and the hydrosmotic action of antidiuretic hormone. The ultrastructural modifications of the epithelium observed under the influence of serosal hypertonicity (the intercellular spaces are dilated while the tight junctions remain closed) lead us to assume that the pathways of water movement across the epithelium could be the same in this case and in hydrosmotic response to the hormone. In contrast, when the mucosal medium is made hypertonic, the ultrastructure is differently altered: the intercellular spaces are closed, the tight junctions show small vesicles and numerous large vacuoles appearing in the cytoplasm.     

The permeability of nonhuman primate vaginal epithelium: a freeze-fracture and tracer-perfusion study

The lining of the vaginal mucosa in primates is a keratinized stratified squamous epithelium. As in other structurally similar epithelia, one function of the vaginal epithelium is to provide a barrier between the external environment and the underlying tissues. The vaginal lining is aglandular and the source of true vaginal fluid has been suggested to be the intercellular channels of the epithelium. On the other hand, other structurally similar epithelia have been shown to have a barrier to the movement of water-soluble molecules through these channels. In the present study, we have examined the permeability of rhesus monkey vaginal epithelium to lanthanum and horseradish peroxidase. Both tracer molecules penetrated the intercellular channels in the lower layers of the epithelium, but were excluded from the channels at and above the granular layer. Neither tracer penetrated significantly between cells at the free surface of the epithelium and usually did not penetrate between cells in the upper layers to any degree from the cut edges of the biopsy. These results are consistent with tracer studies in other structurally similar epithelia and strongly suggest that the upper layers of vaginal epithelium present a barrier to the movement of water-soluble molecules through the intercellular channel system. Freeze-fracture analysis of the epithelium revealed gap junctions and desmosomes between cells in the lower layers, but the former disappear in the upper layers. Unlike other keratinizing epithelia that have been described, random intramembranous particles do not disappear from the plasma membranes of the fully differentiated cells. Fracture planes through the upper layers reveal particle-free lamellae in the intercellular spaces, supporting the idea that intercellular lipids may be one of the components that limits the permeability of the intercellular spaces in this epithelium.     

Hydrogen peroxide induces microvilli on human retinal pigment epithelial cells in culture.

We have found that hydrogen peroxide (10^-4 - 10^-2 M) rapidly induces microvilli on separate cells and confluent sheets of human retinal pigment epithelium in culture. t-butyl hydroperoxide and sodium arsenite do not induce microvilli. A role for hydrogen peroxide as an intercellular messenger has previously been proposed in the inflammatory response, in which hydrogen peroxide from phagocytes may signal to vascular endothelial cells. Our observations thus provide a second example of the induction of what may be a physiological response by this potentially toxic agent. In the retina, hydrogen peroxide released from illuminated photoreceptors may elongate the microvilli which extend into the spaces between them. Increased numbers of microvilli and their protrusion further into the photoreceptor layer may enhance various interactions between the two cell types, including the antioxidant functions of the epithelium.     

Passive Intercellular Pathway in Amphibian Epithelia

PHYSIOLOGIC studies of transporting epithelia generally indicate that passive shunts (or “leak” pathways for water and ions) exist in parallel with transport systems. Most notably, Ussing^1–3 defines this pathway as an extracellular channel in amphibian skin and has shown that a hypertonic external bath decreases the transepithelial electrical resistance, whereas a hypertonic internal bath has the opposite effect. Similar results have been obtained with toad urinary bladder⁴, but in virtually all of the epithelia studied by electron microscopy, tight junctions⁵ have been found at the luminal end of intercellular spaces. Apparent fusion of adjacent plasma membranes and the inability of electron-dense tracer molecules to pass through such regions^5–8 suggest that they may be tight seals, preventing extracellular transepithelial flow. It is shown that these junctions are reproducibly altered when electrical resistance is changed by hypertonic solutions.     

Calcium uptake into guinea-pig trachealis: The effect of epithelium removal

Removal of the epithelium from preparations of guinea-pig airways increases the responsiveness of the smooth muscle of normal and ovalbumin-sensitized animals to a number of contractile agents. To determine if epithelium removal results in an increase in Ca²⁺ entry into the smooth muscle, the effect of removing the epithelium on Ca²⁺ uptake into the trachealis smooth muscle was studied using a modified La³⁺-technique. KCl increased Ca²⁺ uptake in the presence and absence of the epithelium in control and sensitized animals. Methacholine did not promote Ca²⁺ uptake, whether or not the epithelium was present, in either control or sensitized animals. Ovalbumin did not stimulate Ca²⁺ uptake into the trachealis of sensitized animals. These results indicate that the increase in responsiveness of airway smooth muscle seen on epithelium removal is not a consequence of a facilitation of Ca²⁺ entry into the muscle. The increased responsiveness to methacholine in control animals, and to ovalbumin in preparations in tension studies in epithelium-free tissues from sensitized animals, cannot be explained by an increased availability of extracellular Ca²⁺ into the muscle, but, rather may reflect some other effect of the epithelium-derived modulatory factor.     

ULTRASTRUCTURAL STUDIES OF VASOPRESSIN EFFECT ON ISOLATED PERFUSED RENAL COLLECTING TUBULES OF THE RABBIT

Isolated cortical collecting tubules from rabbit kidney were studied during perfusion with solutions made either isotonic or hypotonic to the external bathing medium. Examination of living tubules revealed a reversible increase in thickness of the cellular layer, prominence of lateral cell membranes, and formation of intracellular vacuoles during periods of vasopressin-induced osmotic water transport. Examination in the electron microscope revealed that vasopressin induced no changes in cell structure in collecting tubules in the absence of an osmotic difference and significant bulk water flow across the tubule wall. In contrast, tubules fixed during vasopressin-induced periods of high osmotic water transport showed prominent dilatation of lateral intercellular spaces, bulging of apical cell membranes into the tubular lumen, and formation of intracellular vacuoles. It is concluded that the ultrastructural changes are secondary to transepithelial bulk water flow and not to a direct effect of vasopressin on the cells, and that vasopressin induces osmotic flow by increasing water permeability of the luminal cell membrane. The lateral intercellular spaces may be part of the pathway for osmotically induced transepithelial bulk water flow.     

Epithelial water transport in a balanced gradient system.

The relationship between epithelial fluid transport, standing osmotic gradients, and standing hydrostatic pressure gradients has been investigated using a perturbation expansion of the governing equations. The assumptions used in the expansion are: (a) the volume of lateral intercellular space per unit volume of epithelium is small; (b) the membrane osmotic permeability is much larger than the solute permeability. We find that the rate of fluid reabsorption is set by the rate of active solute transport across lateral membranes. The fluid that crosses the lateral membranes and enters the intercellular cleft is driven longitudinally by small gradients in hydrostatic pressure. The small hydrostatic pressure in the intercellular space is capable of causing significant transmembrane fluid movement, however, the transmembrane effect is countered by the presence of a small standing osmotic gradient. Longitudinal hydrostatic and osmotic gradients balance such that their combined effect on transmembrane fluid flow is zero, whereas longitudinal flow is driven by the hydrostatic gradient. Because of this balance, standing gradients within intercellular clefts are effectively uncoupled from the rate of fluid reabsorption, which is driven by small, localized osmotic gradients within the cells. Water enters the cells across apical membranes and leaves across the lateral intercellular membranes. Fluid that enters the intercellular clefts can, in principle, exit either the basal end or be secreted from the apical end through tight junctions. Fluid flow through tight junctions is shown to depend on a dimensionless parameter, which scales the resistance to solute flow of the entire cleft relative to that of the junction. Estimates of the value of this parameter suggest that an electrically leaky epithelium may be effectively a tight epithelium in regard to fluid flow.     

Ultrastructural study of the effects of 17 beta-oestradiol on the lateral prostate and seminal vesicle of the castrated guinea pig

Administration of oestradiol to castrated animals induced hypertrophy of the secretory cells in the lateral prostate and seminal vesicle. In the lateral prostate, increases in the number of small highly electron-dense granules, multivesicular bodies and intercellular spaces were the prevailing effects 2 weeks after oestradiol treatment. There was also an apparent increase in the amount of cytokeratin intermediate filaments. Prolonged oestradiol administration for 4 weeks showed no appreciable changes in the glandular epithelium when compared with 2-week treatment. However, an increase in the thickness of the fibromuscular layer was observed. In the seminal vesicle, basal cell hyperplasia was associated with a concurrent increase in the size of intercellular spaces 2 weeks after oestradiol administration. There were also apparent increases in the volume of the lamina propria and in the number of stromal cells. An apparent increase in the density of collagen fibres in the stroma was observed 2 and 4 weeks after oestradiol administration. In conclusion, the responses of the epithelium of the lateral prostate and seminal vesicle to a pharmacological dose of oestradiol are different. Prolonged oestradiol administration exerts a more prominent effect on the smooth muscle in the lateral prostate but not in the seminal vesicle. The effects of oestradiol may be mediated directly or indirectly through the other hormones.     

The ultrastructure of the posterior midgut caecum of Pachygrapsus crassipes (Decapoda, Brachyura) adapted to low salinity

The effects of salinity adaptation and of composition and tonicity of fixatives upon the ultrastructure of the posterior midgut caecum (PMC) of Pachygrapsus crassipes have been studied. The PMC epithelium consists of a single layer of columnar cells with a microvillous border. The apical cytoplasm contains numerous mitochondria, lysosomes, and much smooth endoplasmic reticulum. Rough endoplasmic reticulum and Golgi apparatus are situated in the perinuclear cytoplasm. This epithelium resembles other transporting epithelia in that the basal cytoplasm has an extensive system of branched tubules formed from invaginations of the lateral and basal plasma membrane. Numerous mitochondria are associated with the basal tubular system. To determine the possible contribution of the PMC to the osmoregulatory ability of Pachygrapsus, the ultrastructure of the PMC from animals adapted to 40, 50, 100 and 150% sea water was investigated. Enlargement of basal tubules and intercellular spaces at low salinity, suggestive of fluid-transport activity, was found to be an artifact of fixation. The most consistent response when animals were acclimated to dilute salinities was that some basal mitochondria assume a more complex shape, usually appearing as rings in cross sections of the caecum. A hypothesis concerning the functional significance of these mitochondria is proposed.     

Ultrastructural study of the esophagus of seawater-and freshwater-acclimated Mugil cephalus (Perciformes,Mugilidae), euryhaline marine fish

The esophagus of the common grey mullet Mugil cephalus shows a single mode of organization that is common to both seawater- and freshwater-acclimated specimens. A short anterior segment is lined by a stratified epithelium with mucous cells; posteriorly, this is progressively replaced by single-layered columnar epithelium. Ultrastructural features of the columnar epithelium are quite similar in the seawater- and freshwater-acclimated specimens. Apical microvillous projections, lamellar structures, and dilated intercellular spaces are observed. The present study suggests that the mullet esophagus is involved in active ion transport and water permeability, both in seawater and in freshwater environments. © 1993 Wiley-Liss, Inc.     

Neuronal systems immunoreactive with antiserum to lamprey gonadotropin-releasing hormone in the brain of Petromyzon marinus

Summary The wall structure of arteriovenous anastomoses in the rabbit ear was investigated. (1) Clusters of epithelioid smooth muscle cells form 3–4 longitudinally oriented plicae. The channel shows a single, irregularly outlined lumen, and its wall is very thin between adjacent plicae. (2) Endothelial cells covering the plicae protrude into the lumen, thus suggesting active contraction or shortening of the plicae. (3) The tunica adventitia is composed of 4–6 sheaths of flat fibroblasts, which may serve as a barrier to prevent loss of neurotransmitters. Processes of some of the fibroblasts also extend into the tunica media. (4) The tunica media is composed of an outer circular layer of typical smooth muscle cells, and an inner longitudinally running plica of ramified smooth muscle cells. Wide intercellular spaces between these ramified cells are filled with collagen fibrils, microfibrils, amorphous intercellular substances, and fibroblasts. Fibroblasts form close membrane contacts with each other, and with the smooth muscle cells. (5) Fibroblasts and other connective tissue components may function as an elastic support during active motility of the anastomotic channel.