Search PubMed⌕ Search

Biomedical subjects

D R DiBona

Publications and source records attributed to D R DiBona.

At least 19 recordsLinked to original sources
PubMed1987

Intracellular solute gradients during osmotic water flow: an electron-microprobe analysis.

In an attempt to quantify possible intracellular water activity gradients during ADH-induced osmotic water flow, we employed energy dispersive X-ray microanalysis to thin, freeze-dried cryosections obtained from fresh, shock-frozen tissue of the toad urinary bladder. The sum of all detectable small ions (Na + K + Cl) in the cellular water space was taken as an index of the intracellular osmolarity. Presuming that all ions are osmotically active, they comprise about 90% of the cellular solutes. When the cells were exposed to dilute serosal medium, the reduction in the sum of the ions agreed well with the expected reduction in osmolarity. After inducing water flow by addition of ADH and dilution of the mucosal medium, all epithelial cells showed a fall in osmolarity. The change was more pronounced in granular cells than in basal or mitochondria-rich cells, consistent with the notion that granular cells represent the main transport pathway. Most significantly, intracellular osmolarity gradients, largely caused by an uneven distribution of K and Na, were detectable in granular cells. The gradients were not observed after ADH or mucosal dilution alone, or when the direction of transepithelial water flow was reversed. We conclude from these results that there is a significant cytoplasmic resistance to water flow which may lead to intracellular gradients of water activity. Concentration gradients of diffusible cations can be explained by a flow-induced Donnan-type distribution of fixed negative charges. With regard to transepithelial Na transport, the data suggest that ADH stimulates transport by increasing the Na permeability of the apical membranes of granular cells specifically.

Animals↗
PubMed1987

Sulfate transport in apical membrane vesicles isolated from tracheal epithelium.

Sulfate uptake in apical membrane vesicles isolated from bovine tracheal epithelium is shown to occur into an osmotically sensitive intravesicular space, via a carrier-mediated system. This conclusion is based on three lines of evidence: 1) saturation kinetics; 2) substrate specificity; and 3) inhibition by the anion transport inhibitors SITS and DIDS. The affinity of the transport system is highest in low ionic strength media (apparent Km = 0.13 mM) and decreases in the presence of gluconate (apparent Km = 0.68 mM). Chloride appears to cis-inhibit sulfate uptake and to trans-stimulate sulfate efflux. Cis-inhibition and trans-stimulation studies with a variety of anions indicate that this exchange system may be shared by HCO3-, S2O3(2-), SeO4(2-), and MoO4(2-) but not by H2PO4- or HAsO4(2-). Studies indicate that protons may play two distinct roles in sulfate transport in this system. 1) Their possible modifier role is suggested by the fact that protons affect SO2-4 transport in an uncompetitive manner. 2) The possibility that the proton gradient may act as an energy source for a secondary active transport is indicated by the fact that the imposition of a proton gradient stimulates a transient movement of sulfate in to the tracheal apical membrane vesicle, against its concentration gradient, causing an "overshoot" phenomenon. Our studies show that the carrier-mediated system can function in the absence of chloride. The overshoot observed in the presence of a proton gradient (OH- gradient) indicates that under those conditions the mechanism of transport may be a SO4(2-)-OH- exchange. The fact that chloride cis-inhibits and trans-stimulates SO4(2-) transport indicates that SO2-4 uptake may also occur via a SO4(2-)-Cl- exchange. Studies carried out so far do not enable us to conclude unequivocally whether the tracheal apical membrane system displays two distinct carrier activities (SO4(2-)-Cl-; SO4(2-)-OH-) or one anion exchanger, which like the erythrocyte anion exchanger, may interact with SO4(2-), Cl-, and H+. The fact that the anion transport inhibitors DIDS and SITS inhibit SO4(2-) transport in the presence or absence of chloride suggests that the latter possibility may be the case.

Animals↗
PubMed1987

Cell volume regulation in rabbit proximal straight tubule perfused in vitro.

Volume regulation in the perfused proximal nephron of the rabbit was examined quantitatively with a computer-based method for estimating cell volume from differential interference-contrast microscopic images of isolated nephron segments. Following a hyperosmotic challenge (290-390 mosmol), the cells shrank as simple osmometers without a subsequent regulatory volume increase. Conversely, cell swelling induced by a hyposmotic challenge (290-190 mosmol) was completely reversed with a triphasic time course in which a rapid (less than 2 min) initial volume decline was followed by secondary swelling and shrinking phases. A similar regulatory volume decrease was observed following isosmotic cell swelling that was induced by exposure to 290 mosmol, urea-containing solutions. In addition, the cells partially reversed isosmotic swelling that was induced by the luminal replacement of a relatively impermeant cation (i.e., choline) with Na+ and a concomitant increase in luminal solute entry. Our results support two conclusions. First, there exist quantitative differences between the volume regulatory behaviors of perfused and nonperfused proximal tubules, the latter of which exhibit an incomplete and monotonic reversal of hyposmotic cell swelling (M. Dellasega and J. Grantham, Am. J. Physiol. 224: 1288-1294, 1973). Second, the primary physiological role of cell volume regulation in the proximal nephron may be to minimize isosmotic cell swelling associated with acute imbalances in the rates of cell solute entry and exit.

Animals↗
PubMed1987

Regulatory volume decrease in perfused proximal nephron: evidence for a dumping of cell K+.

We utilized the microscopic and morphometric procedures described in the preceding paper to examine the role of a swelling-activated dumping of K-salt in the reversal of hyposmotic cell swelling in the perfused proximal nephron. The rate of the regulatory volume decrease that follows cell swelling in dilute solutions was reduced by two maneuvers that attenuate the K+ chemical potential difference across the basolateral membrane; inhibiting the Na+-K+ pump (e.g., with ouabain) and raising the peritubular K+ concentration. The rate of the regulatory volume decrease was also inhibited by peritubular quinine, which blocks K channels and volume regulation for a number of mammalian cells. Additionally, exposure to hyposmotic solutions resulted in a sustained and quinine-sensitive increase in the apparent permeability of the basolateral membrane to K+ salt, which was monitored qualitatively as the rate of cell volume change that was induced by a perturbation in the peritubular K+ concentration. The simplest interpretation of these results is that the reversal of hyposmotic cell swelling in the proximal nephron is referable at least in part to a swelling-activated loss of K-salt and water from the cells.

Animals↗
PubMed1986

Analysis of structural changes during hypotonic swelling in Ehrlich ascites tumor cells.

Studies were undertaken to quantify structural changes associated with swelling of Ehrlich ascites tumor cells in hypotonic medium. Cells transferred from isotonic (294 mosmol/kg H2O) to hypotonic (98 mosmol/kg H2O) medium swelled rapidly. Subsequently, approximately 40% of the water initially gained was lost, a phenomenon referred to as volume-regulatory decrease (VRD). During the initial rapid cell swelling, blister-like protrusions or blebs were formed on the cell surface. These blebs were examined by routine light microscopy, differential interference-contrast (DIC) microscopy, and scanning and transmission electron microscopy. Microscopic observations and the distribution of ATPase antibodies indicated that the blebs were formed from plasma membrane. During VRD, the blebs coalesced to form a smooth but expanded membrane surface that appeared to be separated from the original cytoplasm by a layer of less dense ground substance. Computer-assisted morphometry from digitized DIC images of the initial swelling phase indicated that all of the volume gained was sequestered in the blebs. We suggest that bleb formation may allow increases in cell volume without disruption of cytoplasmic organization and may be a protective response to a variety of stressful stimuli. The subsequent VRD is accompanied by reduction of this expanded compartment.

Animals↗
PubMed1985

Functional analysis of tight junction organization.

The functional basis of tight junction design has been examined from the point of view that this rate-limiting barrier to paracellular transport is a multicompartment system. Review of the osmotic sensitivity of these structures points to the need for this sort of analysis for meaningful correlation of structure and function under a range of conditions. A similar conclusion is drawn with respect to results from voltage-clamping protocols where reversal of spontaneous transmural potential difference elicits parallel changes in both structure and function in much the same way as does reversal of naturally occurring osmotic gradients. In each case, it becomes necessary to regard the junction as a functionally polarized structure to account for observations of its rectifying properties. Lastly, the details of experimentally-induced junction deformation are examined in light of current theories of its organization; arguments are presented in favor of the view that the primary components of intramembranous organization (as viewed with freeze-fracture techniques) are lipidic rather than proteinaceous.

Animals↗
PubMed1985

Microscopic investigation of structure and function in living epithelial tissues.

In this paper we shall illustrate the utility of direct microscopic methods for studying living epithelia. Beginning with an exposition on the available strategies for visualization of unstained biological materials, the rationale that leads to the choice of differential interference-contrast optics for examination of epithelia is illustrated. Findings from toad urinary bladder, Necturus gallbladder, and rabbit cortical collecting tubule are reviewed. Emphasis on renal structures is provided with a report on work in progress on proximal tubule volume regulation and on structural examination of the isolated perfused macula densa. Conclusions are drawn with respect to the advantages and shortcomings of discussed optical methods and with respect to the choice of model epithelia.

Animals↗
PubMed1979

Cellular site of gastric acid secretion.

Isolated gastric glands of the rabbit were examined both with differential interference-contrast microscopy and with electron microscopy to describe the morphologic correlates of acid secretion. Stimulation of the glands with histamine resulted in the development of intracellular spaces within the parietal cells. A similar transformation was produced by addition of 1 mM aminopyrine, whether the weak base was added in the presence of normal-K+ (5.4 mM) or high-K+ (108 mM) solutions. The intracellular space was compatible with the expanded canaliculus described in stimulated parietal cells. Confirmation that the space produced by histamine is the site of acid secretion was gained by combining fluorescence and interference-contrast methods in the presence of the dye acridine orange, which displays a pH-dependent metachromasia in its emission spectrum. Human gastrin I resulted in an observable discharge of peptic granules.

Acridine Orange↗
PubMed1979

Distribution of Na+-pump sites in transporting epithelia.

There are three techniques for the localization of intraepithelial Na+, K+-ATPase (usually equated with the Na+-pump) that offer reasonable specificity and resolution: the nitrophenylphosphatase assay of Ernst, the immunoferritin procedure of Kyte, and the radioautographic localization of tritiated ouabain as developed by Stirling. These have now been applied to a wide range of epithelia covering the four classes of interest here: isotonic and hypertonic absorbers and isotonic and hypertonic secretors. A review of published results reveals that in every case (except for the choroid plexus) the enzyme is preferentially located on the basolateral surface of the transporting epithelial cells so that a simple correlation of structure and function in terms of the Koefoed-Johnsen and Ussing hypothesis does not seem possible. With little dispute that this enzyme is, nonetheless, the probable site for conversion of metabolic energy to transport-related work, we summarized as well the more macroscopic structural characteristics of epithelia which serve to typify each of the four classes in terms of the direction and tonicity of transported fluid. The apparently systematic differences in cell shape and cell-cell junctions that are summarized here may well be an important consideration for the development of a useful holistic theory with which to explain the transepithelial transport of salt and water.

4-Nitrophenylphosphatase↗
PubMed1978

Pathways for movement of ions and water across toad urinary bladder. III. Physiologic significance of the paracellular pathway.

Hypertonicity of the mucosal bathing medium increases the electrical conductance of toad urinary bladder by osmotic distension of the epithelial "tight" or limiting junctions. However, toad urine is not normally hypertonic to plasma. In this study, the transmural osmotic gradient was varied strictly within the physiologic range; initially hypotonic mucosal bathing media were made isotonic by addition of a variety of solutes. Mucosal NaCl increased tissue conductance substantially. This phenomenon could not have reflected soley an altered conductance of the transcellular active transport pathway since mucosal KCl also increased tissue conductance, whether or not Na+ was present in the bathing media. The effect of mucosal NaCl could not have been mediated solely by a parallel transepithelial pathway formed by damaged tissue since mucosal addition of certain nonelectrolytes also increased tissue conductance. Finally, the osmotically-induced increase in conductance could not have occurred soley in transcellular transepithelial channels in parallel with the active pathway for Na+, since the permeability to 22Na from serosa to mucosa (s to m) was also increased by mucosal addition of NaCl; a number of lines of evidence suggest that s-to-m movement of Na+ proceeds largely through paracellular transepithelial pathways. The results thus establish that the permeability of the limiting junctions is physiologically dependent on the magnitude of the transmural osmotic gradient. A major role is proposed for this mechanism, serving to conserve the body stores of NaCl from excessive urinary excretion.

Animals↗
PubMed1978

Direct visualization of epithelial morphology in the living amphibian urinary bladder.

Differential interference-contrast microscopy has been applied to the study of amphibian urinary bladders, in vitro. It is demonstrated that well-resolved images can be obtained with little loss of tissue viability. Direct observations have been made on the structure of microvilli, the distribution of mitochondria in the mitochondria-rich cells, and the patency of lateral intercellular spaces. It is noted that the effective viscosity of cytoplasm is very high--that it is apparently a gel in which there is no Brownian movement of organelles. The frequency, shape, and pattern of distribution of granular and mitochondria-rich cells is determined for the commonly studied varieties of Bufo marinus. Bladders from Colombian toads contain more and larger mitochondria-rich cells than do those of the Dominican variety. There is no specific arrangement of cell-cell contacts to suggest a structural basis for cooperativeness of action. Finally, a longitudinal study of osmotically-induced changes in the structure of the "tight" or "limiting" junctions establishes the validity of previous findings by electron microscopy.

Animals↗
PubMed1978

Energetics of sodium transport in toad urinary bladder.

The ratio of the rate of transepithelial sodium transport, JNa, across the isolated toad urinary bladder to the simultaneously measured rate of transport-dependent metabolism, JsbCO2, has been measured as a function of the transepithelial electrical voltage, deltapsi. The ratio remains constant with a mean value of 18 to 20 over the range of imposed voltages of 0 to +70 mV. With increasing hyperpolarization of the bladder, JNa decreases and the calculated electromotive force or apparent "ENa" of the sodium pump increases. From thermodynamic and kinetic arguments it is shown that the apparent "ENa" approaches the maximal electrochemical potential gradient, ENa, against which sodium can be transported by this tissue only when JNa approximately 0. At this unique condition F ENa (in which F is the Faraday constant) is the maximal free energy of the chemical reaction driving sodium transport and thus equal to the maximal extramitochondrial phosphorylation potential and the maximal free energy of the mitochondrial respiratory chain within the transporting cells.

Animals↗
PubMed1977

Localization of Na+-pump sites in frog skin.

The localization of Na+-pump sites (Na+-K+-ATPase) in the frog skin epithelium was determined by a freeze-dry radioautographic method for identifying [3H]ouabain-binding sites. Ventral pelvic skins of Rana catesbeiana were mounted in Ussing chambers and exposed to 10(-6) M [3H]ouabain for 120 min, washed in ouabain-free Ringer's solution for 60 min, and then processed for radioautography. Ouabain-binding sites were localized on the inward facing (serosal) membranes of all the living cells. Quantitative analysis of grain distribution showed that the overwhelming majority of Na+-pump sites were localized deep to the outer living cell layer, i.e., in the stratum spinosum and stratum germinativum. Binding of ouabain was correlated with inhibition of Na+ transport. Specificity of ouabain binding to Na+-K+-ATPase was verified by demonstrating its sensitivity to the concentration of ligands (K+, ATP) that affect binding of ouabain to the enzyme. Additional studies supported the conclusion that the distribution of bound ouabain reflects the distribution of those pumps involved in the active transepithelial transport of Na+. After a 30-min exposure to [3H]ouabain, Na+ transport declined to a level that was significantly less than that in untreated paired controls, and analysis of grain distribution showed that over 90% of the ouabain-binding sites were localized to the inner cell layers. Furthermore, in skins where Na+ transport had been completely inhibited by exposure to 10(-5) M ouabain, the grain distribution was identical to that in skins exposed to 10(-6) M. The results support a model which depicts all the living cell layers functioning as a syncytium with regard to the active transepithelial transport of Na+.

Adenosine Triphosphatases↗
PubMed1977

On the distribution of Na+-pump sites in the frog skin.

Exposure of the outside of the isolated frog skin to a Ringer's solution, made hypertonic by the addition of mannitol, causes a rapid and sustained increase in transepithelial permeability through a structural distortion-a focal blistering-of the "tight" junctions of the outermost living cell layer. [(3)H]ouabain, used as an autoradiographic marker for the Na+-pump (Na+-K+-adenosine triphosphatase), is usually unable to penetrate the frog skin from the outside solution, but when added to a hypertonic mannitol- Ringer's solution in the outside bath it readily penetrates the epithelium, presumably through the opened shunt pathway. Radioautographic analysis of [(3)H]ouabain binding sites revealed that most of ouabain enters from the outside solution binds to the sites on the cell membranes of the stratum spinosum, as was the case when it was applied from the inside bath in an earlier study. The outer living cell layer, the first to be exposed to ouabain, does not appear to be the major site for the Na+-pump, and therefore, is not likely to be responsible for most of the active pumping of Na+. This result demonstrates that previous failure to show a high density of Na+-pump sites on the cells of the outermost layer, when [(3)H]ouabain was applied from the inside solution, was not due to the inability of the marker to reach these cells at a sufficient concentration to reveal all pump sites. These results provide further support for a model of Na+-transport across the frog skin which distributes the active pump step on the inward facing membranes of all living cells.

Adenosine Triphosphatases↗
PubMed1976

Role of the septate junction in the regulation of paracellular transepithelial flow.

A comparison of the distribution of septate junctions in invertebrate epithelia and tight junctions in vertebrate systems suggests that these structures may be functionally analogous. This proposition is supported by the internal design of each junction which constitutes a serial arrangement of structures crossing the intercellular space between cells to effectively provide resistance to the paracellular flow of water and small molecules. We have tested the validity of such an analogy by examining whether the osmotic sensitivity of the septate junctions of planarian epidermis follow the rather striking pattern observed for the junctions of very tight vertebrate epithelia (e.g. toad urinary bladder). It has been found that the septate junctions in this system respond in similar fashion to their vertebrate counterparts, blistering with accumulated fluid when the medium outside the epidermis is made hypertonic with small, water-soluble molecules. We conclude that the two types of junction probably are functionally analogous and that, in each case, this rectified structural response to transepithelial osmotic gradients may be indicative of the role of such structures in the transport function of epithelia.

Animals↗
PubMed1976

The protective effect of hyperosmotic mannitol in myocardial ischemia and necrosis.

Morphologic and hemodynamic changes that occur following coronary occlusion are examined. The effectiveness of hyperosmotic mannitol in lessening the extent of myocardial damage is assessed and mechanisms for its action discussed. Forty and 60 min of coronary vascular occlusion followed by 15 and 45 min of reflow were associated with a persistence of ischemia following reflow of blood, as established by infusions of silastic into the aortic root. Electron microscopic studies demonstrated myocardial and endothelial cell swelling at the end of the reflow period. The process of cell swelling appeared to be initiated during the period of arterial occlusion. This cell swelling was reduced by elevation of serum osmolality by 30-40 mOsm above control with the administration of mannitol during and following occlusion. There was an associated 40-50% reduction of vascular resistance following occlusion if mannitol was administered. In addition, the extent of necrosis, which was widespread in untreated hearts 12 hours after occlusion, was strikingly less in the hearts of dogs which received mannitol. Thus, in ischemic myocardium, elevation of osmolality by mannitol reduces myocardial necrosis, probably through its restoration of normal cell volume.

Animals↗