Biomedical subjects

D C Dawson

Publications and source records attributed to D C Dawson.

At least 55 records · Page 3Linked to original sources

PubMed1986

Differentiation of two distinct K conductances in the basolateral membrane of turtle colon.

The K conductance of the basolateral membrane of turtle colon was measured in amphotericin-treated cell layers under a variety of ionic conditions. Changing the composition of the bathing solutions changed not only the magnitude but also the physical properties of the basolateral K conductance. The results are consistent with the notion that altered ionic environments can lead to changes in the relative abundance of two different populations of K channels in the basolateral membrane, which can be differentiated on the basis of pharmacological specificity, ion selectivity, and tracer kinetics. In the following article (Germann, W. J., S. A. Ernst, and D. C. Dawson, 1986, Journal of General Physiology, 88:253-274), we present evidence consistent with the hypothesis that one of these conductances was due to the same channels that give rise to the normal resting basolateral K conductance of the transporting cells, while the other was associated with experimental maneuvers that led to extreme swelling of the epithelial cells.

Amphotericin B↗

PubMed1986

Resting and osmotically induced basolateral K conductances in turtle colon.

Two types of K conductance can be distinguished in the basolateral membranes of polyene-treated colonic epithelial cells (see Germann, W. J., M. E. Lowy, S. A. Ernst, and D. C. Dawson, 1986, Journal of General Physiology, 88:237-251). The significance of these two types of K conductance was investigated by measuring the properties of the basolateral membrane under conditions that we presumed would lead to marked swelling of the epithelial cells. We compared the basolateral conductance under these conditions of osmotic stress with those observed under other conditions where changes in cell volume would be expected to be less dramatic. In the presence of a permeant salt (KCl) or nonelectrolyte (urea), amphotericin-treated colonic cell layers exhibited a quinidine-sensitive conductance. Light microscopy revealed that these conditions were also associated with pronounced swelling of the epithelial cells. Incubation of tissues in solutions containing the organic anion benzene sulfonate led to the activation of the quinidine-sensitive gK and was also associated with dramatic cell swelling. In contrast, tissues incubated with an impermeant salt (K-gluconate) or nonelectrolyte (sucrose) did not exhibit a quinidine-sensitive basolateral conductance in the presence of the polyene. Although such conditions were also associated with changes in cell volume, they did not lead to the extreme cell swelling detected under conditions that activated the quinidine-sensitive gK. The quinidine-sensitive basolateral conductance that was activated under conditions of osmotic stress was also highly selective for K over Rb, in contrast to the behavior of normal Na transport by the tissue, which was supported equally well by K or Rb and was relatively insensitive to quinidine. The results are consistent with the notion that the basolateral K conductance measured in the amphotericin-treated epithelium bathed by mucosal K-gluconate solutions or in the presence of sucrose was due to the same channels that are responsible for the basolateral K conductance under conditions of normal transport. Conditions of extreme osmotic stress, however, which led to pronounced swelling of the epithelial cells, were associated with the activation of a new conductance, which was highly selective for K over Rb and was blocked by quinidine or lidocaine.

Amphotericin B↗

PubMed1986

Single potassium channels blocked by lidocaine and quinidine in isolated turtle colon epithelial cells.

The patch-clamp technique for recording single-channel currents across cell membranes was applied to single turtle colon epithelial cells isolated with hyaluronidase. With electrodes fabricated from Corning #7052 glass, high-resistance seals were consistently formed to these cells. In on-cell patches with low K (2.5 mM) in the pipette and high K (114.5 mM) in the bath, outward K currents were recorded that had a slope conductance of 17 pS and a reversal potential greater than -70 mV. Currents through this K channel were blocked by lidocaine, quinidine, and barium. These agents also block a cell swelling-induced K conductance identified by macroscopic current measurements in the basolateral membranes of the intact colonic epithelium, suggesting that the 17 pS K channel identified by single-channel recording in isolated turtle colon cells may be responsible for this macroscopically defined K conductance.

Animals↗

PubMed1986

Cholinergic regulation of Na absorption by turtle colon: role of basolateral K conductance.

The mechanism underlying the muscarinic inhibition of colonic Na absorption is unknown. In this study the effects of carbachol on active Na transport and basolateral K conductance were compared in the isolated turtle colon. Carbachol produced a biphasic response in both Na transport and basolateral K conductance. The response consisted of a transient activation followed by a sustained inhibition and was blocked by atropine. Submucosal cholinergic neurons were implicated in the regulation of colonic transport by employing depolarizing agents to release endogenous acetylcholine. Depolarizing agents produced a carbachol-like response that was atropine-sensitive. Finally, experiments with the Ca ionophores, A23187 and ionomycin, suggested that the muscarinic response may be mediated, at least in part, by changes in cellular Ca. These experiments provide evidence that cholinergic neurons are present in the turtle colon submucosa, muscarinic agonists cause a change in basolateral K conductance that may be an important event in the regulation of colonic Na absorption, and a Ca second messenger system may be involved in mediating the response.

Animals↗

PubMed1986

K+ transport by rat colon: adaptation to a low potassium diet.

Recent studies with the isolated perfused rat kidney have demonstrated the existence of an intrinsic renal adaptation to conserve K+ in response to ingestion of a low K+ diet for 3 days. To determine whether the colon alters its K+ transport properties in a similar fashion, we measured transmural 86Rb fluxes across sheets of distal colonic epithelium under short-circuit conditions. Preliminary studies using a double-isotope technique demonstrated that 86Rb and 42K fluxes were similar; therefore 86Rb flux was considered equivalent to K+ flux. The distal half of the colon from each rat was divided into two segments, referred to as early and late distal colon. Experiments were carried out using rats fed a K+ -free, control (0.15 mmol/g), and high K+ (1.13 mmol/g) powdered diet of otherwise identical electrolyte content. Net K+ secretion (Jnet) by the early distal colon was reduced from 0.45 in the controls to -0.02 mueq X cm-2 X h-1 by a low K+ diet as a result of a decrease in serosal-to-mucosal flux (Jsm), with no change in mucosal-to-serosal flux (Jms). Conductance (GT) and short-circuit current (Isc) were unchanged. Jnet by the late distal colon averaged 0.17 in the controls and 0.01 mueq X cm-2 X h-1 with a low K+ diet, but this difference was not significant statistically. In comparison with the controls, a high K+ diet had no effect on Jnet by the early distal colon (0.48 mueq X cm-2 X h-1) but increased Jnet by the late distal colon substantially (0.77 mueq X cm-2 X h-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Physiological↗

PubMed1985

Passive cation permeability of turtle colon: evidence for a negative interaction between intracellular sodium and apical sodium permeability.

The role of intracellular sodium in the regulation of apical sodium permeability was investigated in an electrically "tight" epithelium, the turtle colon. In the presence of low mucosal sodium (3 mM) and serosal ouabain, an inhibitor of the basolateral sodium pump, the apical membrane retained a substantial amiloride-sensitive, sodium conductance and the basolateral membrane exhibited a barium-sensitive potassium conductance in parallel with a significant sodium (and lithium) conductance. In the presence of a high mucosal sodium concentration (114 mM), however, inhibition of active sodium absorption by ouabain led to a disappearance of the amiloride-sensitive, transepithelial conductance that was due, at least in part, to a virtual abolition of the apical sodium permeability. Two lines of evidence indicate that this permeability decrease was dependent upon an increase in intracellular sodium content. First, raising the mucosal sodium concentration from 3-114 mM in the presence of ouabain reversibly inhibited the amiloride-sensitive conductance. The time course of the decline in conductance paralleled the apparent intracellular accumulation of sodium in exchange for potassium, which was monitored as a transient deflection in the amiloride-sensitive, short-circuit current. Second, the inhibitory effect of mucosal sodium-addition was markedly attenuated by serosal barium, which prevented the accumulation of sodium by blocking the electrically coupled, basolateral potassium exit. These results support the notion of a "negative feedback" effect of intracellular sodium on the apical sodium permeability.

Amiloride↗

PubMed1985

Aldosterone does not stimulate the Na:K pump in isolated turtle colon.

The study of the mechanisms by which mineralocorticoids stimulate sodium absorption across distal epithelia has focused on three possible sites of action: apical sodium permeability, the basolateral Na:K pump, and the production of high-energy substrates. Recently we developed a method for direct measurement of the current generated by the basolateral Na:K pump of the turtle colon [15]. In the presence of mucosal amphotericin-B and serosal barium the short-circuit current across the colon can be equated with the current produced by active electrogenic exchange of sodium for potassium across the basolateral membrane. This pump current is a measure of the transport capacity of the epithelial Na:K pump that is uncomplicated by changes in apical membrane sodium permeability. Pump currents, thus defined, were compared in control tissues and tissues treated with aldosterone in vitro. After 9 h Na absorption was increased 4-fold in the aldosterone-treated tissues but the values of the pump current were identical in the two groups. This result indicates that acute stimulation of sodium absorption by aldosterone does not occur by stimulating the Na:K pump directly.

Aldosterone↗

PubMed1985

Primary culture of duck salt gland. I. Morphology of confluent cell layers.

Dissociated avian salt gland secretory cells were maintained in primary culture after plating on hydrated collagen gels. When seeded at 3 X 10(6) cells/cm2, confluent cell sheets formed within 2-3 days, whereas cultures seeded at lower densities formed a complex reticulum of cell aggregates, which remained nonconfluent even after 7 days. Scanning electron microscopy showed that the free surface of 3-day confluent cultures consisted of intermixed convex and flattened cell membranes with prominent junctional boundaries and abundant microvilli. Transmission electron microscopy indicated that these cultures were multilayers of 1-4 cells in thickness. The plasma membranes of the superficial cells were polarized into apical and basolateral regions displaying, respectively, microvilli and interdigitating lateral membrane folds. These membrane domains were separated by shallow occluding junctions, which consisted of both single strands and simple net-like arrays in freeze-fracture images. Underlying epithelial cells retained lateral membrane folds and formed desmosomal contacts with superficial and neighboring cells. These cultures, unlike the intact tissue, allow direct access to the apical and basolateral cell surfaces for electrophysiological analysis of transmural active ion transport.

Animals↗

PubMed1985

Primary culture of duck salt gland. II. Neurohormonal stimulation of active transport.

Primary cultures of structurally polarized sheets of avian salt gland secretory cells were mounted in Lucite chambers for transmural electrophysiological analysis. Transmural resistance values increased during the first 3 days of culture to 293 +/- 35 omega X cm2 and then decreased slowly thereafter. There was little short-circuit current (Isc) in the absence of secretagogues. Serosal addition of either carbachol or epinephrine resulted in a Isc consistent with positive charge flow from mucosa to serosa, thus demonstrating that these cell layers were capable of active ion transport in response to either cholinergic or adrenergic neurohormonal stimulation. Serosal ouabain or furosemide abolished the response to either agonist, while theophylline enhanced the response. Receptor specificity for the electrical responses was shown by selective inhibition of carbachol- and epinephrine-induced Isc by atropine and propranolol, respectively. The results demonstrate that these primary epithelial cell cultures are capable of active ion transport and are sensitive to known inhibitors of secretory transport, and suggest that intracellular coupling mechanisms for hormonal control are retained in culture. These cultures should be useful for studying mechanisms of ion secretory transport and their regulatory control.

Amphotericin B↗

PubMed1984

Potassium transport by turtle colon: active secretion and active absorption.

Measurement of transepithelial potassium fluxes in the absence of transmural electrochemical potential gradients showed that the isolated turtle colon can actively absorb and actively secrete K+. Under short-circuit conditions the active secretory flow was inhibited by mucosal amiloride, whereas the absorptive flow was unaffected by the diuretic. The effects of ouabain and barium on secretory flow were consistent with a simple model involving basolateral uptake by an Na+-K+-ATPase and conductive exist across the apical and basolateral membranes. The active absorptive flux was blocked by mucosal ouabain and by serosal barium. The opposing active flows clearly represented cellular K+ transport, whereas paracellular K+ flows behaved as expected for a free-solution shunt.

Absorption↗

PubMed1984

Control of potassium transport by turtle colon: role of membrane potential.

To more clearly define the role of the transepithelial electrical potential difference (V m----s), potassium permeability, and sodium-potassium pump rate in transcellular potassium transport by isolated turtle colon, we measured transmural potassium fluxes under open-circuit conditions in the presence and absence of putative blockers of potassium transport: amiloride and barium. The results were consistent with the notion that V m----s is a major determinant of cellular potassium secretion, whereas active potassium absorption is insensitive to changes in V m----s. These observations suggest that "coupling" between colonic sodium absorption and potassium secretion in vivo could be due primarily to the effect of the lumen negative V m----s on transcellular secretory potassium flow. Amiloride-induced inhibition of potassium secretion appeared to be due to the reductions in V m----s and sodium-potassium pump rate that accompanied the inhibition of active sodium absorption.

Amiloride↗

PubMed1983

Basolateral potassium channel in turtle colon. Evidence for single-file ion flow.

Treatment of the apical surface of the isolated, ouabain-inhibited turtle colon with the polyene antibiotic amphotericin B permitted the properties of a barium-sensitive potassium conductance in the basolateral membrane to be discerned from the measurements of transepithelial fluxes and electrical currents. Simultaneous measurements of potassium currents and 42K fluxes showed that the movement of potassium was not in accord with simple diffusion. Two other cations, thallium and rubidium, were also permeable and, in addition, exhibited strong interactions with the potassium tracer fluxes. The results indicate that permeant cations exhibit positive coupling, which is consistent with a single-file mechanism of ion translocation through a membrane channel.

Animals↗

PubMed1983

Mechanism of epithelial lithium transport. Evidence for basolateral Na:Na and Na:Li exchange.

Measurement of transmural sodium fluxes across isolated, ouabain-inhibited turtle colon in the presence of a serosal-to-mucosal sodium gradient shows that in the absence of active transport the amiloride-sensitive cellular path contains at least two routes for the transmural movement of sodium and lithium, one a conductive path and the other a nonconductive, cation-exchange mechanism. The latter transport element can exchange lithium for sodium, and the countertransport of these two cations provides a mechanistic basis for the ability of tight epithelia to actively absorb lithium despite the low affinity of the basolateral Na/K-ATPase for this cation.

Animals↗

PubMed1980

Active sodium transport by turtle colon via an electrogenic Na-K exchange pump.

Active sodium absorption by a variety of epithelia is abolished by ouabain, but the obligatory coupling between the movement of sodium and potassium expected from a basolateral (Na+ + K+) ATPase has not been convincingly demonstrated. According to the model of Koefoed-Johnsen and Ussing, the asymmetric cation selectivities of the apical and basolateral membranes prevent basolateral Na-K exchange from being expressed as opposing transmural ion flows. An additional consequence of this asymmetry is that the short-circuit current (Isc) cannot be identified with the current through the sodium-potassium pump. We used the polyene antibiotic, amphotericin-B, to reduce the resistance and the cation selectivity of the apical membrane of isolated turtle colon so that the basolateral membrane current could be dissected into two components: one through a barium-sensitive potassium channel and another which represents the current associated with ouabain-sensitive, electrogenic, Na-K exchange. Comparison of cation fluxes and short circuit current indicates that in these conditions active sodium absorption is entirely attributable to an electrogenic Na-K pump with a stoichiometry of approximately 3Na:2K.

Amphotericin B↗

PubMed1978

Sodium uptake across the apical border of the isolated turtle colon: confirmation of the two-barrier model.

The initial rate of Na uptake by the turtle colon from the mucosal bathing solution consists of two operationally distinct components. One component is a linear function of mucosal Na concentration, is unaffected by amiloride, and appears to represent Na uptake into the paracellular shunt path. The major component of Na uptake is abolished by amiloride and is virtually equal to the short-circuit current over a wide range of mucosal Na concentrations, suggesting that this portion of Na uptake represents Na movement into Na-transporting cells of the colon. The amiloride-sensitive component of Na uptake, at low mucosal Na concentrations, was unaffected if net Na transport was abolished by ouabain. Similarly, at low mucosal Na concentrations the amiloride-sensitive conductance of the colon was identical in the presence and in the absence of net Na transport. These results show that the isolated turtle colon behaves as two distinct barriers to transmural Na transport, an apical barrier blocked by amiloride and a more basal-lying barrier where active, transmural Na transport is blocked by ouabain. In addition, these experiments appear to provide the first unambiguous demonstration that the initial-rate isotope uptake technique can provide a direct measure of the properties of the amiloride-sensitive barrier to transmural Na movement, presumably the apical membranes of the Na-transporting cells. The results are consistent with the notion that the rate of transmural active Na transport and the conductance of the active Na-transport path are determined by the properties of the apical membrane.

Amiloride↗

PubMed1978

Induction of reverse flow of Na+ through the active transport pathway in toad urinary bladder.

When active Na+ transport across the toad urinary bladder was abolished by ouabain, a 'reversed' short circuit current could be induced by an Na+ concentration gradient. This reversed current was increased by vasopressin and inhibited by amiloride and appears to represent net Na+ movement 'backwards' through epithelial cells which normally participate in active Na+ transport across the bladder.

Amiloride↗

PubMed1978

Cation selectivity of the apical membrane of the turtle colon: sodium entry in the presence of lithium.

Exposure of the apical surface of the isolated turtle colon to Li produced a marked transient in short-circuit current (ISC) and total tissue conductance (GT) which was abolished by amiloride but was unaffected by ouabain or by removing Na or Cl from the mucosal bathing solution. Despite marked changes in Isc, Na uptake across the apical membrane was a linear function of time during exposure to Li-containing solutions, and except at very high Li concentrations, the initial rate of Na uptake, JiNa, was identical to its pre-Li value. In the presence of Li, however, JiNa was significantly less than the total Isc. The apparent "transference number" for Na in the apical membranes was a function of the Li:Na concentration ratio in the mucosal bathing solution. These results suggest that Li can carry substantial amounts of current through amiloride-sensitive channels in the apical membrane of the colon without having any effect on the rate coefficient for Na entry. This behavior is not consistent with "competition" of Na and Li for a membrane "carrier" but rather suggests that the Na entry mechanism may be a population of pores or channels through which Na and Li may pass with negligible interaction.

Amiloride↗

PubMed1978

Transport characteristics of isolated newborn rabbit ileum.

Segments of ileum from newborn rabbits were mounted as flat sheets in Lucite chambers and transmural fluxes of Na, Cl, and alanine were measured in the absence of electrochemical potential gradients. In the presence of 140 mM Na, the ileum of the newborn exhibited a serosa-positive electrical PD and a corresponding short-circuit current (Isc) which was markedly enhanced by the addition of D-glucose of L-alanine to the mucosal solution. Alanine-induced increments in Isc were a saturable function of the mucosal alanine concentration. In the presence of Na, alanine was actively absorbed, and the net alanine flux was a saturable function of alanine concentration. When the Na in the bathing solutions was completely replaced by choline, Isc declined to near zero, and the response of Isc to mucosal alanine was abolished. In addition, active amino acid transport was abolished in the absence of Na. In the absence of alanine, the isolated ileum of the newborn actively absorbed Na and Cl, and the algebraic sum of the net movements of these ions accounted for Isc. In the presence of alanine, active Cl transport was abolished and Isc was equal to the net Na absorption. These results indicate that the ileum of the newborn rabbit is similar to that of the adult in its ability to actively absorb Na, Cl, and alanine, but differs from the ileum of the adult by having a greater passive permeability to ions and amino acid, and differs particularly with regard to the effect of mucosal alanine on transmural ion transport.

Alanine↗