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Biomedical subjects

A D Macknight

Publications and source records attributed to A D Macknight.

At least 37 records · Page 2Linked to original sources
PubMed1984

Effects of medium acetate on cellular volume in rabbit renal cortical slices.

Slices of rabbit renal cortex were incubated at 25 degrees C in media in which acetate replaced chloride. There was gross cellular swelling in isosmotic 132 mM-acetate medium, and this swelling was unique in that, with a normal medium potassium concentration, it was accompanied by a substantial increase in cellular potassium content. This accumulation of potassium, but not the cellular swelling, was dependent upon metabolism and inhibited by ouabain. This accumulation of potassium was not dependent upon the cellular swelling. It also occurred in a hyperosmotic acetate medium in which swelling was minimized. In isosmotic media, the cellular swelling was proportional to medium acetate concentration and was also affected markedly by medium pH, being greatest at an initial medium pH of 7.1 and least at pH 7.7. The swelling was reversed and cellular composition restored when tissue was re-incubated in NaCl medium. Ouabain (10(-3)M) largely prevented this recovery in volume. The results are consistent with plasma-membrane-based theories, on the assumption that membranes are much more permeable to undissociated acetic acid than they are to the acetate ion. They are inconsistent with the expectations of an alternative hypothesis (the association--induction hypothesis) which ascribes the maintenance of cellular composition to properties of cellular proteins and cellular water rather than to those of the plasma membrane. The results do not favour the suggestion that cellular swelling itself results in irreversible cellular damage. The results are consistent with the hypothesis that the ouabain-inhibitable Na-K-ATPase plays a major role in the regulation of cellular volume. No alternative metabolically dependent volume regulating mechanism need be postulated to explain them.

Acetates↗
PubMed1983

Intra- and transepithelial analytical techniques.

Epithelia transport a variety of solutes and water. Study of such transport requires a determination of the driving forces responsible for transport, of the pathways through which transport occurs, and of the factors controlling such transport. Transepithelial driving forces are readily determined where the composition of the bathing media can be altered and electrical forces negated. Where substances move only through a paracellular pathway such manipulations may be adequate to define the permeability and selectivity of the pathways. For substances utilizing a cellular pathway, driving forces and permeabilities across the two dissimilar apical and basolateral cellular membranes must be determined. Where a substance can be shown to move across a membrane against its electrochemical potential gradient, the source of the energy for such movement must be assessed. This review focuses on the applicability and validity of a variety of techniques utilized for the study of epithelial transport to answer these questions. These include microelectrode techniques, chemical analyses, microprobe analysis, microscopy, and techniques for assessing the coupling of metabolism to transport.

Animals↗
PubMed1982

Cellular lithium and transepithelial transport across toad urinary bladder.

Toad urinary bladders were exposed on either their mucosal or serosal surfaces, or on both surfaces, to medium in which sodium was replaced completely by lithium. With mucosal lithium Ringer's, serosal sodium Ringer's, short-circuit current (SCC) declined by about 50 percent over the first 60 min and was then maintained over a further 180 min. Cellular lithium content was comparable to the sodium transport pool. With lithium Ringer's serosa, SCC was abolished over 60 to 120 min whether the mucosal cation was sodium or lithium. Measurements of cellular ionic composition revealed that the epithelial cells gained lithium from both the mucosal and serosal media. With lithium Ringer's mucosa and serosa, cells lost potassium and gained lithium and a little chloride and water, but these changes in cellular ions could not account for the current flow across the tissue under these conditions, which must, therefore, have been carried by a transepithelial movement of lithium itself. The inhibition by serosal lithium of SCC was overcome by exposure of the mucosal surface of the bladders to amphotericin B. Thus it reflected, predominantly, an inhibition of lithium entry to the cells across the apical membrane. It is suggested that this inhibition is a consequence of cellular lithium accumulation.

Amiloride↗
PubMed1982

Ions and water in the epithelial cells of rabbit descending colon.

1. Isolated sheets of rabbit descending colon epithelial cells stripped from their underlying muscle coats were incubated in chambers at 37 degrees C with oxygenated media, and their non-inulin space water, sodium, potassium and chloride contents were subsequently determined. 2. With sodium Ringer bathing both surfaces, amiloride, 10(-4) M, decreased non-inulin space sodium content by 76 mmol/kg dry wt. Ouabain, 10(-3) M, caused loss of non-inulin space potassium which was not completely compensated for by uptake of sodium over 30 min incubation. Chloride and water, therefore, decreased. Amiloride, 10(-4) M, inhibited but did not prevent this uptake of sodium after ouabain. 3. Tissues exposed to sodium-free choline Ringer rapidly exchanged non-inulin space sodium for choline and, more slowly, lost potassium, chloride and water. The equilibration of sodium in the non-inulin space when sodium Ringer was restored to the mucosal medium alone was largely amiloride-insensitive. For restoration of non-inulin space potassium to normal levels, sodium was required in the serosal but not the mucosal medium. 4. Neither the absence of glucose nor the absence of chloride from the mucosal medium affected the non-inulin space sodium content when sodium was restored to the mucosal medium bathing sodium-depleted tissues. 5. It is argued that, whereas non-inulin space potassium and water contents are synonymous with their cellular values, only about one third of non-inulin space sodium is cellular when sodium Ringer bathes both surfaces, and the concentration of the sodium within the cellular transport pool approximated 20 mmol/kg H2O, consistent with estimates obtained from other techniques.

Amiloride↗
PubMed1982

Alternative methods for measurement of membrane potentials in epithelia.

Methods for the measurement of membrane potentials in cells not easily penetrated by microelectrodes were assessed for use in epithelia. The lipophilic cation triphenylmethylphosphonium appeared to distribute in a Nernstian fashion in the epithelial cells of Necturus gallbladder as judged by parallel microelectrode measurements. In the toad urinary bladder, the distribution of this cation gave a value for the membrane potential of epithelial cells under short-circuit conditions of -62 mV in normal Ringer's solution and -51 mV after 2 h treatment with ouabain. In our laboratory the dye 3,3'-dipropylthiadicarbocyanine iodide, when used with cell suspensions, yielded results comparable to those of other workers, but we were unable to record a redistributional signal from epithelial sheets or scraped cells. The dye appeared to enter cells and become irreversibly bound. The membrane-bound merocyanine dyes have not been used on epithelial cells. They appear to hold the greatest promise for dynamic experiments on epithelial membrane potentials.

Animals↗
PubMed1981

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

The structural consequences of clamping the transepithelial potential difference across the toad's 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.

Amiloride↗
PubMed1981

Structural responses to voltage-clamping in the toad urinary bladder. II. Granular cells and the natriferic action of vasopressin.

The natriferic action of vasopressin has been investigated with morphological studies of voltage-clamped toad urinary bladders. Granular cell swelling can be induced in the presence of isoosmotic solutions when the orientation of the transmural potential is reversed by voltage clamping (V.A. Bobrycki, J. W. Mills, A.D.C. Macknight & D. R. DiBona, J. Membrane Biol., 60:21, 1981) and results from an increased rate of sodium entry across the mucosal membrane; under these conditions the active transport mechanism at the basal-lateral membrane becomes rate-limiting. Vasopressin exacerbated the voltage-reversal-induced swelling of granular cells while other cell types were unaffected. Granular cell swelling appeared to be dependent upon sodium entry from the mucosal medium since it was completely prevented by amiloride. There was no evidence for an effect of vasopressin on tight junction permeability; voltage-reversal induced the same amount of junction blistering whether or not vasopressin was present. It is concluded that the predominant effect of vasopressin on transepithelial sodium transport is to increase the sodium conductance of the mucosal plasma membrane. As is the case with the hydroosmotic effect of the hormone, the natriferic action of vasopressin seems to be exerted primarily, if not entirely, on the granular cells.

Amiloride↗
PubMed1981

Interaction of ouabain with the Na+ pump in intact epithelial cells.

To determine the specificity and efficacy of [(3)H]ouabain binding as a quantitative measure of the Na(+) pump (Na(+), K(+)-ATPase) and as a marker for the localization of pumps involved in transepithelial Na(+)-transport, we analyzed the interaction of [(3)H]ouabain with its receptor in pig kidney epithelial (LLC-PK(1)) cells. When these epithelial cells are depleted of Na(+) and exposed to 2 muM [(3)H]ouabain in a Na(+)-free medium, binding is reduced by 90 percent. When depleted of K(+) and incubated in a K(+)- free medium, the ouabain binding rate is increase compared with that measured at 5 mM. This increase is only demonstable when Na(+) is present. The increased rate could be attributed to the predominance of the Na(+)-stimulated phosphorylated form of the pump, as K(+) is not readily available to stimulate dephosphorylation. However, some binding in the K(+)-free medium is attributable to pump turnover (and therefore, recycling of K(+)), because analysis of K(+)-washout kinetics demonstrated that addition of 2 muM ouabain to K(+)-depleted cells increased the rate of K(+) loss. These results indicate that in intact epithelial cells, unlike isolated membrane preparations, the most favorable condition for supporting ouabain binding occurs when the Na(+), K(+)-ATPase is operating in the Na(+)-pump mode or is phosphorylated in the presence of Na(+). When LLC-PK(1) cells were exposed to ouabain at 4 degrees C, binding was reduced by 97 percent. Upon rewarming, the rate of binding was greater than that obtained on cells kept at a constant 37 degrees C. However, even at this accelerated rate, the time to reach equilibrium was beyond what is required for cells, swollen by exposure to cold, to recover normal volume. Thus, results from studies that have attempted to use ouabain to eliminate the contribution of the conventional Na(+) pump to volume recovery must be reevaluated if the exposure to ouabain was done in the cold or under conditions in which the Na(+) pump is not operating.

Animals↗
PubMed1980

Comparison of analytic techniques: chemical, isotopic, and microprobe analyses.

The development of new techniques and modifications to existing methods are providing important information about cellular water and electrolytes in relation to transepithelial transport. Information obtained for one epithelium, the toad urinary bladder, from chemical analyses, isotopic studies, and electron microprobe analysis, is reviewed. Chemical and isotopic analyses allow estimation of average cellular water and ion contents and determination of the origin of ions from the media bathing the mucosal and basolateral cellular membranes. The electron microprobe provides additional information about the distributions of ions between the cell types that constitute the epithelial layer and also allows some measurement of ion distributions within individual cells. In toad urinary bladder epithelial cells there is broad agreement between the results obtained with these different techniques and a summary is provided of possible relationships between transepithelial sodium transport and cellular composition.

Animals↗
PubMed1979

Localization of [3H]ouabain-sensitive Na+ pump sites in cultured pig kidney cells.

Pig kidney cells, LLC-PK1, grown by standard tissue-culture techniques form monolayers and maintain morphological features characteristic of epithelia. Cultures exposed to 2 X 10(-6) M [3H]ouabain for 30 min at 37 degrees C bound 7.77 +/- 0.37 pmol/mg protein. This could be reduced by 58% by incubation in the presence of 45 mM K+. Freeze-dry radioautographic localization of [3H]ouabain-binding sites revealed grains distributed only along that fraction of the plasmalemma directly facing the culture-dish surface. Binding and localization of [3H]ouabain were correlated with an inhibition of the Na+ pump in these cells because analysis of cellular electrolytes in control cultures versus those exposed to 10(-3) M ouabain revealed a fall in K+ from 419 +/- 9 to 173 +/- 4 mmol/kg dry wt with a reciprocal increase in Na+. There was no change in cell H2O. Similarly, oxygen consumption was reduced by 32% after exposure to ouabain. These results provide direct evidence that in epithelial cells in culture the membrane facing the culture dish corresponds to the basolateral membrane of epithelial cells in vivo.

Animals↗
PubMed1978

Electron microprobe analysis of the different epithelial cells of toad urinary bladder. Electrolyte concentrations at different functional states of transepithelial sodium transport.

The electrolyte composition of toad urinary bladder epithelial cells has been measured using the technique of electron microprobe analysis. Portions of hemibladders, which had been mounted in chambers and bathed with a variety of media, were layered with albumin solution on their mucosal surfaces and immediately shock-frozen in liquid propane at -180 degrees C. From the frozen material 1--2 micrometer thick cryosections were cut and promptly freeze-dried for 12 hr at-80 degrees C and 10(-6) Torr. Electron microprobe analysis using a scanning electron microscope, an energy dispersive X-ray detector, and a computer programme, to distinguish between characteristic and uncharacteristic radiations, allowed quantification of cellular ionic concentrations per kg tissue wet wt by comparison of the intensities of the emitted radiations from the cells and from the albumin layer. Granular, mitochondrial-rich, and basal cells, and the basal portions of goblet cells, showed a similar composition, being high in K (about 110 mM/kg wet wt) and low in Na (about 13 mM/kg wet wt). The apical portions of goblet cells were higher in Ca and S and lower in P and K, presumably reflecting the composition of the mucus within them. With Na-Ringer's as the mucosal medium, cells gained Na and lost K, when their serosal surfaces were exposed to ouabain, 10(-2) M. Replacement of mucosal Na by choline virtually prevented these ouabain-induced changes. Cellular ion contents were unchanged when Na in the serosal medium was replaced by choline. No differences in Na and K concentrations were detected between nuclei and cytoplasm. These results provide independent support for the hypothesis the the cellular Na transport pool in toad bladder epithelial cells derives exclusively from the mucosal medium and that no important recycling of Na occurs from the serosal medium to the cells.

Animals↗
PubMed1978

Epithelial cell electrolytes in relation to transepithelial sodium transport across toad urinary bladder.

Aspects of the relationships between cellular composition and transepithelial sodium transport across toad urinary bladder are reviewed. Changes in cellular sodium produced by amiloride, vasopressin, aldosterone, hypoxia, ouabain, and sodium-free media are consistent with a cellular sodium transport pool. Metabolic studies suggest that this pool gains its sodium from the mucosal medium and that there is little recycling of sodium between cell and serosal medium. One-third of the cellular potassium equilibrates readily with serosal potassium. The rate of exchange of potassium is much less than the rate of sodium transport supporting the contention that sodium transport in this tissue is electrogenic. Studies with 36Cl suggest that chloride does not cross the apical cellular membranes, but exchanges with serosal chloride. Possible relationships between transepithelial sodium transport and cellular volume regulation are discussed.

Aldosterone↗
PubMed1978

The sodium transport pool.

The sodium transport pool in epithelial cells represents sodium involved in active transport across the epithelium. There has been much controversy about the size of such a pool and even about its existence. Techniques for estimating the size of this pool are described. By analysis of toad bladder epithelial cells scraped from hemibladders mounted in chambers under a variety of conditions it has proved possible to detect and to quantify a sodium transport pool. Only about 20 percent of non-inulin space sodium measured flame photometrically is contained in this pool. This represents the total sodium entering cells from the mucosal medium, is in good agreement with the cellular sodium measured by the electron microprobe, and averages some 10-16 mmol/kg tissue H2O. Measurements of the pool in other tissues are considered.

Animals↗
PubMed1977

Contribution of mucosal chloride to chloride in toad bladder epithelial cells.

Epithelial cells were scraped from the bladders of toads of the species Bufo marinus obtained from the Dominican Republic. These epithelial cells exchanged their chloride virtually completely with 36Cl i; the medium within 60 min. Of this chloride, about 93% came from the serosal medium. The approximately 20 mmole/kg dry wt of chloride which equilibrates with 36Cl in the mucosal medium was still present when choline replaced sodium in the medium in the presence of amiloride (10(-4) M) and was almost all readily removed by rapid washing of the mucosal surface immediately prior to analysis. These observations suggest that little chloride of mucosal origin is truly intracellular. This conclusion is supported by the fact that after vasopressin the increased cellular chloride was not of mucosal origin.

Animals↗
PubMed1977

Effects of replacing medium sodium by choline, caesium, or rubidium, on water and ion contents of renal cortical slices.

1. Renal cortical slices from rat, rabbit, and guinea-pig were incubated in media in which choline, caesium or rubidium replaced sodium.2. Slices of rabbit and guinea-pig renal cortex incubated in oxygenated choline Ringer decreased in volume initially and did not swell over 3 hr at 25 degrees C. There was a steady loss of potassium. Inhibition of metabolism (N(2) + 1 mM iodoacetamide) caused some swelling. Ouabain, 10 mM, in choline Ringer affected neither loss of potassium nor tissue water content.3. Slices of rat renal cortex similarly incubated in choline Ringer swelled over 3 hr at 25 degrees C whether or not metabolism was inhibited; ouabain (15 mM) affected neither tissue potassium loss nor tissue water content.4. Incubation in choline Ringer containing either 0.2 mMp-chloromercuribenzoic acid, or 1 mM ethacrynic acid increased the tissue water content of guinea-pig renal cortical slices.5. Depletion of cellular potassium (by preliminary incubation in oxygenated potassium-free sodium Ringer with 10 mM ouabain at 30 degrees C) resulted in increased tissue water content when rabbit renal cortical slices were subsequently incubated in oxygenated choline Ringer at 25 degrees C for 3 hr.6. There was no evidence of energy-dependent extrusion of water or ions from either equilibrated rat or rabbit renal cortical slices leached at 0.5 degrees C and then reincubated at 25 degrees C in choline Ringer.7. Rat and guinea-pig renal cortical slices leached at 0.5 degrees C and reincubated at 25 degrees C swelled in rubidium Ringer and in caesium Ringer. There was no evidence of energy-dependent water or ion extrusion when metabolism was restored after leaching in either of these media. Metabolizing rat slices but not guinea-pig slices swelled faster than slices whose metabolism was inhibited.8. These results lend no support to the mechano-chemical hypothesis which ascribes cellular volume regulation to a contractile mechanism squeezing isotonic extracellular fluid from the cells. Instead it is suggested that cellular water content in these experiments reflects the balance between the rate of loss of potassium (and chloride) from the cells and the rate of uptake of extracellular cation (and chloride) into the cells - these rates reflecting both the electrochemical potential gradients of the ions and membrane permeability to them. The implications in relation to the hypothesis of ouabain-insensitive cellular volume regulation are discussed.

Animals↗
PubMed1977

Transepithelial sodium transport and carbon dioxide production by the toad urinary bladder in the absence of serosal sodium.

1. The production of CO2 in relation to sodium transport by toad urinary bladder has been examined in the absence of sodium from the serosal medium. 2. Replacement of serosal sodium by choline increased CO2 production without stimulating short-circuit current. Replacement of serosal medium by Tris did not have this effect. 3. With serosal sodium Ringer replaced by Tris Ringer, the ratio of sodium transported to CO2 produced was not altered significantly. 4. The results therefore suggest that there is no important recycling of sodium between the serosal medium and the transporting epithelial cells.

Animals↗