Donald R. DiBona

The cellular specificity of the effect of vasopressin on toad urinary bladder.

SummaryPhase and electron micrographs of toad bladders were obtained following dilution of bathing media in the presence and absence of vasopressin. Dilution of the mucosal medium alone resulted in no morphologic changes. Subsequent addition of vasopressin produced an increase in the cell volume of the granular cells, manifested by some or all of the following changes: increased area of granular cell profiles as observed in sections, rounding of the cell nucleus, displacement of the two components of the nuclear envelope, loss of nuclear heterochromatin, sacculation of the endoplasmic reticulum and the Golgi apparatus, and reduction in the electron density of the cell cytoplasm. No such morphologic changes were noted in the other cell types comprising the mucosal epithelium — the mitochondria-rich, the goblet, and the basal cells. On the other hand, dilution of the serosal bathing medium in the absence of vasopressin caused a marked increase in the cell volume of all these cell types. The results demonstrate that the action of vasopressin to enhance bulk water flow across toad bladder is exerted specifically on the apical surface of the granular cells. It is suggested that the hormonal effect on sodium transport may also be limited to the granular cells. The route of osmotic water flow and the possible role of the other mucosal epithelial cells is discussed.

Pathways for movement of ions and water across toad urinary bladder: I. Anatomic site of transepithelial shunt pathways

SummaryMucosal hypertonicity, produced by the addition of NaCl, KCl, mannitol, urea, sucrose or raffinose, reduced the electrical resistance of toad urinary bladder and induced bullous deformations (blisters) of the most apical junctions of the mucosal epithelium: the smaller solutes were most effective in eliciting both phenomena. Study of the effect of addition and subsequent removal of mannitol from the mucosal medium indicated that both the electrical and morphologic changes are reversible and follow the same time course. Mucosal hypertonicity induced comparable changes in the tissue in the presence or absence of inhibition of active sodium transport by replacement of sodium by choline, or by addition of ouabain or amiloride. Dilution of the tonicity of the serosal medium similarly reduced the tissue resistance and induced blisters within the epithelium, demonstrating that the osmotic gradient, rather than the mucosal hypertonicity itself is the cause of the osmotically-induced resistance change. The data indicate, therefore, that the osmotic gradient reduces the electrical resistance of the tissue primarily by deforming the apical junctions.The simplest interpretation of the data is that the apical tight junctions are considerably more permeable to water and small solutes than had previously been thought. Addition of solute to the mucosal medium leads to the diffusion of solute into the junctions: the subsequent transfer of water from the lateral intercellular spaces and/or the adjacent cellular cytoplasm, deforms these structures and reduces the resistance to the passage of ions across the tissue. The results suggest that the apical junctions constitute the rate-limiting permeability barrier of the putative parallel shunt pathway across toad bladder.

Measurement of the composition of epithelial cells from the toad urinary bladder.

SummaryTwo methods are described by which epithelial cells from toad urinary bladders can be obtained for analysis of their intracellular water and electrolyte contents. In the first, a method similar to that described in 1968 by J. T. Gatzy and W. O. Berndt, sheets of epithelial cells are scraped from bladders after incubation in sodium Ringers and collagenase (400 mg/liter). The scraped cells were incubated under various conditions and their composition subsequently determined. Oxygen consumption was also measured. In the second method, epithelial cells were scraped from hemibladders removed from chambers. These cells were then analyzed without further incubation. The morphology of epithelial cells obtained by each method is illustrated. Both methods yield similar results and evidence is provided that the derived intracellular values obtained truly reflect the composition of the epithelial cells.

A Study of Intercellular Spaces in the Rabbit Jejunum during Acute Volume Expansion and after Treatment with Cholera Toxin

The effects of acute volume expansion and of intraluminal administration of cholera toxin have been examined in rabbit jejunum. Acute volume expansion was shown to reverse the normal reabsorptive flux of water and cause significant fluid secretion. Phase and electronmicroscopic examination of the jejunal epithelium showed that marked distension of the intercellular spaces had occurred. Examination of the jejunal epithelium after treatment with cholera toxin showed that, in association with high rates of fluid secretion, the intercellular spaces were extremely small and lateral membranes of adjacent cells were in close apposition to one another. Thus the mechanisms of fluid secretion in these two situations would appear to be quite different. The secretion associated with volume expansion, and accompanied by a rise in venous pressure and bullous deformations of terminal junctions, could well be due to hydrostatic pressure applied through intercellular channels. The secretion of cholera appears to be unrelated to hydrostatic pressure and is more likely due to body-to-lumen active ion transport.

Cell volume regulation and ischemic tissue damage

Over years of friendly meetings with Professor Aharon Katzir-Katchalsky, many topics of mutual interest were discussed. He was the ideal person to come to with a problem. After being subjected to his critical, analytic mind, most research problems seemed simple, more clearly defined and understandable. His broad biologic and scientific background grew from an apparently insatiable interest in all natural phenomena. He generously shared his knowledge and imparted his wisdom with a share of his own infectious excitement. He was quick to sense the significance of understanding of biological processes to their practical application. For this reason it seems appropriate to relate the progress made in the understanding of cell volume regulation, which had been discussed on several occasions with him, to its possible significance as a factor in disease processes.

Structural responses to voltage-clamping in the toad urinary bladder. I. The principal role of granular cells in the active transport of sodium.

SummaryThe structural consequences of clamping the transepithelial potential difference across the toads urinary bladder have been examined. Reducing the potential to zero (short-circuiting) produced no apparent changes in the morphology of any of the four cell types which comprise the epithelium. Computer assisted, morphometric analysis of quick frozen specimens revealed no measurable difference in granular cell volume between open- and short-circuited preparations. However, when the open-circuit potential was quantitatively reversed (serosa negative with respect to mucosa), some of the preparations showed a marked increase in granular cell volume. To examine this more systematically twelve preparations were voltage-clamped at 50 mV (serosa negative); eight of the twelve revealed prominent granular cell swelling relative to control, short-circuited preparations. Only in this group of eight had the external circuit current fallen substantially during the clamping interval. Mitochondria-rich cells were not affected detectably. Application of the diuretic amiloride prior to clamping at reversed potential prevented granular cell swelling in every case. Goblet cells which were often affected by the −50 mV clamp were not protected by the diuretic. Granular cell swelling thus appeared to be dependent on sodium entry at the mucosal surface. We also observed that, after voltage reversal, the apical “tight” junctions of the bladders were blistered as they are with hypertonic mucosal media. This blistering was associated with an increase in passive ionic permeability and was not prevented by application of amiloride. This finding is consistent with the evidence that the junction is a complex barrier with asymetric, and hence, rectifying properties for intrinsic ionic conductance as well as hydraulic permeability. These findings, together with others from the literature, lead to the conclusion that the granular cells constitute the principal, if not sole, elements for active sodium transport across toad urinary bladder and that they swell when sodium entry exceeds the transport capacity of the pump at the basal-lateral surface.

The protection of renal function from ischemic injury in the rat

AbstractThe role of cell swelling in the development of tissue injury was investigated in an ischemic model of acute renal failure in the rat. One kidney was selectively flushed with a 10% solution of polyethylene-glycol M.W. 6,000 (PEG) in normal saline prior to the clamping of the vascular pedicle, and the contralateral kidney was flushed with normal saline. Both kidneys remained ischemic for 45 min with the test solutions within their extracellular space; the concentration of PEG was chosen to prevent cell swelling during the ischemic period.After 24 h of reflow, a striking difference in function was found. The endogenous creatinine clearance in the PEG-treated kidney was 2.41±0.18 ml · min−1 Kg BW−1

Clarification of the intercellular space phenomenon in toad urinary bladder.

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