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Biomedical subjects

R L Duncan

Publications and source records attributed to R L Duncan.

At least 37 records · Page 2Linked to original sources
PubMed1988

Effect of aldosterone on 86Rb fluxes in cultured kidney cells (A6).

This study was designed to evaluate the relative contributions of hormone induced changes in active and passive K+ transport in an epithelial cell line in continuous culture derived from toad kidney (A6) using 86Rb as a tracer for measuring unidirectional K+ fluxes. The effects of 24 h exposure to aldosterone (A) and aldosterone plus insulin (A+I) on unidirectional K+ fluxes were evaluated under short-circuited conditions and under open circuit conditions. In epithelia exposed to A, a small but significant amount of active K+ secretion was found, although it was not significantly greater than in control epithelia. The bidirectional fluxes in both A and A+I treated epithelia, under short-circuited conditions, increased by a similar amount over control values indicating an increase in apparent permeability of passive transepithelial K+ transport. Under open circuit conditions, A stimulated net K+ transport by about 5-fold over controls. The increase in K+ secretion produced by A under open circuit conditions could be explained by the combined effects of an increase in transepithelial K+ permeability and an increase in the transepithelial electrical potential difference (PD). The presence of I produced no additional effects to that of A on K+ transport under the conditions used in this study. It is concluded that the substantial increase in K+ secretion induced in A6 cells by 24 h exposure to A is primarily passive in nature. It is possible that the changes in both PD and transepithelial K+ permeability, which can account for the observed increase in K+ secretion, are secondary to the stimulation of active Na+ transport.

Aldosterone↗
PubMed1987

Demonstration of a direct effect of atrial natriuretic factor on epithelium: study in cultured pig kidney cells.

Cultured renal tubular cells represent a convenient model to assess a possibility of a direct effect of atrial natriuretic factor (ANF) on renal epithelial cells independent of hemodynamic influences. LLC-PK1 cells were grown to confluence on collagen-coated polycarbonate membranes. Studies were performed in modified Ussing chambers with a symmetrical composition of bathing solutions. LLC-PK1 monolayers expressed polarity with transepithelial potential differences (PD) 0.54 +/- 0.31 mV, short-circuit current (SCC) 1.85 +/- 0.49 microA cm-2, and transepithelial resistance 308 +/- 79 omega cm2. ANF caused a decrease in PD and SCC. The effect of ANF was abolished by eliminating Na+ from the bathing solution. Dibutyryl cyclic GMP (db-c-GMP) and Na+ nitroprusside mimicked the effect of ANF, which had no additive effect following pretreatment with these compounds. Cyclic AMP (c-AMP) reversed the effect of ANF. 22Na+ unidirectional flux was significantly inhibited by ANF. In summary, in cultured renal tubular cells ANF decreases SCC and PD, and decreases Na+ influx, suggesting a direct effect on epithelial ion transport.

Animals↗
PubMed1986

The interaction of Escherichia coli with normal human serum: the kinetics of serum-mediated lipopolysaccharide release and its dissociation from bacterial killing.

We have examined the killing of E. coli and kinetics of lipopolysaccharide (LPS) release after the exposure of the bacteria to normal human serum (NHS) and sera deficient in complement components, or with inactivated complement components. LPS of the galactose epimerase-deficient strain E. coli J5 were specifically radiolabeled by growing the bacteria in a medium containing [3H]galactose. Exposure of the washed bacteria to NHS resulted in a significant reduction (greater than 99%) in viability within 15 min and the concomitant release of radiolabeled LPS. However, maximal release of LPS was consistently 30% of the total radiolabel incorporated into the LPS molecules. The amount of tritium-labeled LPS released was shown to be directly proportional to the concentration of bacteria exposed to NHS, suggesting that release of LPS was not limited by the availability of some critical serum component(s). The consumption of complement in NHS by incubation with E. coli was demonstrated by decreased alternative and classical pathway-specific hemolytic activity. The use of Factor D-depleted and VEM-treated human sera demonstrated that, with these bacteria, both the alternative and classical pathways of complement contribute to bacterial killing and release of LPS. It is noteworthy that, in VEM-treated and Factor D-depleted sera, the rate of killing and the kinetics of LPS release were somewhat slower as compared to control serum. Bacterial killing in C7-depleted and C9-deficient human sera was minimal. Neither killing nor LPS release occurred in heat-inactivated (56 degrees C, 30 min) human serum. The amount of [3H]LPS released by C9-deficient serum was qualitatively similar to the amount released by the action of NHS. Tritium-labeled LPS was not released in C7-depleted serum. These data indicate that bacterial killing can be dissociated from LPS release, and suggest that, whereas LPS release may be necessary for the bactericidal effects of serum complement, it is probably not sufficient to effect killing. Furthermore, a significant fraction of LPS can be removed from the outer membrane of the bacteria without an apparent affect on viability.

Adult↗
PubMed1986

Immunologic activity of lipopolysaccharides released from macrophages after the uptake of intact E. coli in vitro.

Lipopolysaccharides (LPS) have been isolated from culture supernatants and from cell lysates after the in vitro phagocytosis of E. coli by murine macrophages. By using E. coli radiolabeled specifically in the LPS component with [3H]galactose, our studies have shown that the macrophage-"processed" LPS is enhanced with respect to its immunostimulatory activity in comparison with control phenol-water-extracted LPS. As assessed by its ability to induce interleukin 1 production in naive macrophages or proliferation in cultures of murine splenocytes, the macrophage-processed LPS is between 10- and 100-fold greater in specific activity. Evidence is presented for both structural and chemical alterations in the LPS macromolecule.

Adjuvants, Immunologic↗
PubMed1985

A corticosterone metabolite produced by A6 (toad kidney) cells in culture: identification and effects on Na+ transport.

A6 cells form typical tight epithelia when grown in culture on permeable supports and exhibit active Na+ transport [short circuit current (Isc)], which is stimulated by aldosterone and corticosterone. Previous studies demonstrated nuclear binding of polar corticosterone metabolites produced by the cells. This study was performed to determine whether sufficient quantities of the metabolite(s) are released into the medium of A6 cells for identification and to test for agonist activity on active Na+ transport. Cells were incubated in [3H]corticosterone (10(-8)-10(-4) M) for 24 h. Approximately 25-35% of the radiolabel, recovered in ethyl acetate extracts of medium, chromatographed on reverse phase HPLC as a single peak more polar than corticosterone. This derivative cochromatographed with 6 beta-hydroxycorticosterone (6 beta-OH-corticosterone) on HPLC and normal phase high performance TLC. Mass spectroscopy of 6 beta-OH-corticosterone and the unknown yielded 10 identical molecular ions, including the molecular ion with a mass to charge ratio of 362 corresponding to the mol wt of 6 beta-OH-corticosterone stimulated Isc in A6 epithelia with a time course typical of a steroid and an EC50 of 10(-6) M. The Isc induced by 6 beta-OH-corticosterone was equivalent to net Na+ flux, indicating active Na+ transport stimulation. At maximum effective concentrations of corticosteroids, 6 beta-OH-corticosterone plus aldosterone induced a greater Isc stimulation than aldosterone alone, suggesting that at least a portion of the effect of 6 beta-OH-corticosterone is mediated by a steroidal pathway other than that used by aldosterone. Also, corticosterone produced twice the Isc increase produced by aldosterone. Thus, 6 beta-OH-corticosterone may contribute to the enhanced corticosterone effect on Isc compared to aldosterone alone.

Aldosterone↗
PubMed1985

In vivo biological activities of endotoxin.

The basic mechanisms by which bacterial lipopolysaccharides (LPS) interact with cells and tissues of the endotoxin sensitive host have been examined within the context of defining critical targets for the manifestation of the multiple pathophysiologic effects of this potent bacterial toxin. Evidence has been presented to suggest that metabolic processing of bacteria by phagocytic cells can result in the release of biologically active endotoxin. The available experimental data would indicate that, in the mouse, a bone marrow derived radiosensitive cell is responsible for the toxic effects of endotoxin. The precise mechanism by which lipopolysaccharides interact with these cells remains to be elucidated. Although interaction with critical targets on the membrane of LPS responsive cells is established, the evidence for specific endotoxin receptor molecules is weak and still controversial. Recent data suggest that, if such endotoxin receptors do, in fact exist, they are at best only weakly immunogenic. The use of the C3H/HeJ "endotoxin unresponsive" mouse strain, however, remains as an extremely useful experimental model to define the mode of action of endotoxin at the molecular level.

Animals↗
PubMed1985

Sodium transport and distribution of electrolytes in frog skin.

The objective of this study on frog skin was to examine correlations between transepidermal active Na-transport and intracellular [Na]c, [K]c, [Cl]c homeostasis. Isolated, whole skins, and "split skins" were used in measurements of short-circuit current (SCC) and open skin potential (PD). Water and ion contents were estimated on split skins. Absolute [Na]c and [K]c varied over the range of 18 to 46, and 113 to 80 mM, respectively (Figure 7), but a complementary relationship existed between Na and K, such that [Na]c + [K]c remained approximately equal to 129 mM. Average values for [Na]c and [K]c were approximately equal to 31 and approximately equal to 96 mM, respectively. [Cl]c remained constant at approximately equal to 38 mM. This complementary relationship does not seem to be an artifact, caused by collagenase, used in the preparation of split skins. Whole skins and split skins in Ringer's solution, when treated with fluoroacetate (FAc), ouabain (Ou), or vanadate (Va) over wide ranges of concentrations, showed that FAc greatly depressed the SCC and the PD, without changing [Na]c, [K]c, [Cl]c. FAc acted only from the corium side of the skin. The decreasing SCC remained a Na-current, as in control skins. By comparison, such a separation of cellular functions could not be established with Ou, or Va. These inhibitors either affected SCC, PD, and cellular ion concentration, or they had no effect on any of these parameters. The complementary relationship between [Na]c and [K]c, with [Cl]c remaining again at approximately equal to 38 mM, was also found in tissues exposed to inhibitors. These results indicate that transcellular active Na transport and electrolyte homeostasis are not always rigidly coupled, suggesting that these processes may not be uniformly distributed within the epithelial cells, or among the interconnected cell layers of the frog skin epidermis.

Animals↗
PubMed1984

The fate of E. coli lipopolysaccharide after the uptake of E. coli by murine macrophages in vitro.

The fate of bacterial lipopolysaccharide (LPS) after the uptake of Escherichia coli by macrophages in vitro was studied. The LPS of the galactose epimerase-deficient E. coli J5 mutant was specifically radiolabeled with [3H]galactose by growing the organism in a basic salts medium containing galactose. Control bacteria were uniformly radiolabeled by growth in [14C]glucose and unlabeled galactose-containing medium. Surface constituents of E. coli were also labeled with 125I. After in vitro phagocytosis of labeled E. coli by murine peritoneal exudate macrophages, the rate of exocytosis of LPS, as assessed by release of 3H over a 72-hr period, was considerably reduced in comparison with other bacterial constituents (14C and 125I release). The [3H]galactose-labeled material exocytosed from macrophages and that remaining intracellularly (obtained from macrophage lysates) were isolated by cesium chloride (CsCl) density gradients and were shown to have altered density profiles as compared with purified E. coli LPS. The macrophage-"processed" [3H] galactose-containing fractions from CsCl density gradients of culture supernatants or macrophage lysates were capable of clotting Limulus amebocyte lysate. The [3H]galactose material obtained from 48-hr macrophage lysates and culture supernatants could also induce a lethal response in actinomycin D-treated mice. These data suggest that bacterial LPS may be selectively retained by the macrophage and that the post-phagocytic events that result in bacterial degradation are not accompanied by the degradation of LPS. Furthermore, although the LPS may be modified by the macrophage, it retains its biologic activity.

Animals↗
PubMed1983

Fungistatic mechanism of human transferrin for Rhizopus oryzae and Trichophyton mentagrophytes: alternative to simple iron deprivation.

Human serum, human transferrin (TF), and the iron chelator 1,10-phenanthroline (OP) produce iron-reversible fungistatic activity which has been attributed to simple iron deprivation. In this study, the influence of the size of the inoculum on the inhibitory activity of serum, TF, and OP prepared with the same iron-binding capacity (2.5 micrograms/ml) for Rhizopus oryzae and Trichophyton mentagrophytes was examined. Inhibition was monitored in liquid microcultures maintained at 37 degrees C and pH 7.4 to 7.5 by measuring the change in absorbance density. Increasing the number of spores in the inoculum disrupted the fungistatic activity of serum and TF, but not that of OP. The dilution at which OP lost fungistatic activity was not affected by the number of spores in the inoculum and was the same for both fungi. The dilution at which TF and serum lost fungistatic activity was dependent upon both the quantity of the inoculum and the species of fungus. The number of viable spores, rather than the total number of spores in the inoculum, was determined to be important in overcoming the inhibition of fungal growth by serum and TF. The fungistatic activity of serum and TF could be diminished by the preexposure of the serum to viable but nongrowing spores. Direct and indirect fluorescence studies indicated that both T. mentagrophytes and R. oryzae absorbed TF. Glucose uptake by R. oryzae was inhibited by a 4-h exposure to 5.0 to 0.15 mg of apotransferrin per ml. These results suggest that the fungistatic activity of TF for R. oryzae and T. mentagrophytes may not be attributable to simple iron deprivation and raise the possibility of a requirement for a direct interaction.

Antifungal Agents↗
PubMed1982

Fungistatic capacity of sera from guinea pigs injected with various iron solutions: differences between Trichophyton mentagrophytes and Rhizopus oryzae.

The fungistatic capacity and serum ion levels (SI) of guinea pigs given subcutaneous injections of various iron solutions were examined. The administration of 2.0 ml of 0.1 M ferric ammonium sulfate, ferric sulfate, or ferric chloride subcutaneously had no significant effect on the SI 3 h after administration, whereas ferric ammonium citrate, ferric citrate, or ferrous sulfate elevated the SI to 50 to 140 times that necessary to saturate the unbound transferrin in normal sera. The sera from 11 of 15 guinea pigs with an elevated SI remained fungistatic for Trichophyton mentagrophytes, whereas 3 of 15 guinea pigs with an elevated SI remained fungistatic for Rhizopus oryzae. The sera from normal guinea pigs were consistently fungistatic for Rhizopus oryzae. The sera from normal guinea pigs were consistently fungistatic for both T. mentagrophytes and R. oryzae. These data suggest that subcutaneous administration of certain iron compounds can significantly elevate the SI without completely abolishing the fungistatic capacity of the serum.

Animals↗
PubMed1978

(1-(3-(Phenothiazin-10-yl)propyl)-4-piperidinyl)phenylmethanones, a novel class of long-acting neuroleptic agents.

In previous studies the phenyl-4-piperidinylmethanone moiety was shown to be a neuroleptic pharmacophore. A short series of [1-[3-(phenothiazin-10-yl)propyl]-4-piperidinyl]phenylmethanones was prepared and tested for neuroleptic activity using the blockade of d-amphetamine lethality in aggregated mice and suppression of conditioned avoidance behavior as the end points. Most compounds were shown to be potent neuroleptic agents and two were found to possess a long duration of action.

Animals↗