Basal membrane uptake in potassium-secreting cells of midgut of tobacco hornworm (Manduca sexta)

1990 ◽  
Vol 258 (1) ◽  
pp. R112-R119
Author(s):  
A. C. Chao ◽  
A. R. Koch ◽  
D. F. Moffett

Basal membrane voltage (Vb), intracellular K+ activity [(K+)i], and short-circuit current (Isc) were measured in isolated posterior midguts of Manduca sexta wherein Isc is a measured of active secretion of K+ from blood into lumen. When bathed in 32 mM K+ and exposed to 100% O2, average values were Isc = 244 microAmp/cm2, Vb = -33.1 mV, and (K+)i = 88.6 mM. The electrochemical gradient across the basal membrane (d mu) averaged +5.8 mV (a gradient favorable for K+ entry). Exposure to 5% O2 led to a new steady state in which Isc = 71 microAmp/cm2, Vb = -18.7 mV, and (K+)i = 99.4 mM. During hypoxia, d mu averaged -9.9 mV (a gradient unfavorable for K+ entry). When the external bathing solution was 10 mM K+, comparable values were, for 100% O2, Isc = 139 microAmp/cm2, Vb = -56.1 mV, (K+)i = 72.2 mM, and d mu = +3.6 mV, and in 5% O2 the values were Isc = 28.3 microAmp/cm2, Vb = -43.7 mV, (K+)i = 76.1 mM, and d mu = -10.2 mV. The failure of cellular K+ to fall during prolonged hypoxia is evidence for a thermodynamically active basal K+ uptake process.

1979 ◽  
Vol 78 (1) ◽  
pp. 213-223
Author(s):  
DAVID F. MOFFETT

Potassium transport by the isolated midgut of Manduca larvae, as measured by the short circuit current, is inhibited by substitution of small organic solutes (M.W. < 340) for the sucrose normally included in bathing solution formulated for this tissue. Other solutes of molecular weight equal to or greater than sucrose are essentially as effective as sucrose in promoting the short circuit current. Equilibration of midgut in solutions containing the small solute mannitol results in a decrease in the dry weight/wet weight ratio of the tissue, suggesting that the small solutes can penetrate into areas of the tissue which are not accessible to sucrose. Histological studies suggest that sites of swelling in the presence of mannitol include both cytoplasm and goblet cell lumen. The inhibition of the short circuit current is rapidly reversible on return to bathing solution containing sucrose or another large solute. The effect of small solutes probably does not involve compromise of the energy source for potassium transport since oxygen uptake is unchanged in the presence of a small solute.


1984 ◽  
Vol 246 (6) ◽  
pp. G732-G744
Author(s):  
M. A. Imon ◽  
J. F. White

Titration techniques and K+- sensitive microelectrodes have been used to investigate the relations among HCO3(-) absorption, luminal K+, and intracellular K+ activity in in vitro Amphiuma jejunum. The HCO3(-) absorptive flux (JHCO3(-] measured by pH-stat under short circuit was reduced by removal of K+ from the medium but not by replacement of Na+ with choline. JHCO3(-) exhibited a seasonal variation when K+ was absent from the media and was increased to a maximum when K+ equaled 5 mM. Addition of K+ to a K+-free luminal medium stimulated JHCO3(-) much more than addition to the serosal medium. Acetazolamide (10(-4) M) blocked K+-stimulated HCO3(-) absorption while benzolamide reduced the short-circuit current associated with HCO3(-) absorption much more rapidly when added to the mucosal bathing medium. Intracellular K+ activity (aik) and mucosal membrane potential (psi m) of jejunal villus cells were measured with double-barreled microelectrodes. When bathed bilaterally with HCO3(-)-containing media, K+ was actively accumulated for many hours (aik = 58.5 mM) but in the presence of ouabain fell to equilibrium (16 mM) after 2 h. In contrast, when HCO3(-) absorption was induced by removal of serosal HCO3(-), aik was elevated to 83.6 mM and, after 4-h exposure to ouabain cell K+, remained far above electrochemical equilibrium at 33 mM. Tissues bathed in Na+-free (Tris) media containing ouabain retained cell K+ after 4 h at even higher levels (46 mM). Cell K+ activity was reduced by removal of K+ from either the mucosal or serosal medium. Acetazolamide reduced aik over 2 h in Na+-free media from 66 to 42 mM. The decline in aik was associated with a concomitant decline in the HCO3(-) absorptive current. It is concluded that K+ is actively accumulated across both luminal and serosal membranes of the jejunal absorptive cell and that the luminal uptake mechanism is linked to HCO3(-) absorption or an equivalent process.


1975 ◽  
Vol 228 (4) ◽  
pp. 1162-1171 ◽  
Author(s):  
JT Gatzy

Fluxes of ions and water across the short-circuited, excised bullfrog lung were determined by radioisotope techniques. The unidirectional flows of Na+, K+, Ca++, TcO4 minus, HCO3 minus, gluconate, rho-aminohippurate, dinitrophenolate, SO4 equal to, and water were symmetrical. Both HCO3 minus fluxes were reduced by acetazolamide. In contrast, Cl minus, Br minus I minus, and SCN minus movement from serosa to mucosa exceeded the flux in the opposite direction. Net Cl minus transport followed the kinetics of a saturable process and was inhibited by dinitrophenol and hypoxia. These results indicate an active secretion of halide anions and SCN minus into the lumen. Attempts to demonstrate Br minus anatagonism of Cl minus transport were equivocal. Cl minus transport accounted for 50 percent minus of the early short-circuit current but after 90 min the two measurements were equal. Incubation of the lung in bicarbonate-free Ringer revealed unequal decreases in the H+ concentration of the bathing solutions. Net "base" addition to the serosal solution was reduced by prior removal of the blood from the pulmonary vasculature. Therefore, "base" release could not be localized to the epithelia. The Na+, K+, Ca++ and Cl minus composition of the lung tissue was unchanged over 3 h. Since tissue and, hence cell Cl minus is lower than the concentration in the bathing solution the Cl pump is probably located in the luminal border of the alveolar epithelial cell.


1991 ◽  
Vol 157 (1) ◽  
pp. 243-256 ◽  
Author(s):  
DAVID F. MOFFETT ◽  
ALAN KOCH

The isolated posterior midgut of the tobacco hornworm maintains a vigorous transepithelial K+ transport from the hemolymphal side to the lumen side at a rate accurately measured by its short-circuit current. Previous studies using the K+ channel blocker Ba2+ suggested that partial inhibition of the short-circuit current by hemolymphal Ba2+ was due to blockage of one of at least two parallel transbasal entry routes for K+ into the intracellular transport pool. The present studies show that the local anesthetic lidocaine, at a concentration of 5 mmoll−1 on the hemolymphal side, partly inhibits net transepithelial K+ transport. The inhibition is accompanied by hyperpolarization of the basal membrane and an increase in transbasal resistance, suggestive of a block of transbasal K+ conductance. The effects of lidocaine and Ba2+ are additive, suggesting that the inhibitors distinguish separate, parallel K+ uptake processes.


1988 ◽  
Vol 255 (2) ◽  
pp. G247-G252 ◽  
Author(s):  
A. N. Charney ◽  
J. I. Scheide ◽  
P. M. Ingrassia ◽  
J. A. Zadunaisky

Chloride absorption in the small intestine of the winter flounder, Pseudopleuronectes americanus, is reported to be sensitive to ambient pH. We studied this sensitivity in isolated stripped intestinal mucosa mounted in modified Ussing chambers. Unidirectional 36Cl fluxes (JClm----s, JCls----m) were measured under short-circuited conditions in bathing solutions containing various combinations of HCO3- (0-20 mM), partial pressure of CO2 (0-36 mmHg), and pH (6.77-7.85). We found that JClm----s, net 36Cl flux (JClnet), and short-circuit current (Isc) increased and JCls----m decreased predominately in response to increases in bathing solution pH. There was a linear relationship between pH and both JClnet (r = 0.92, P less than 0.01) and Isc (r = 0.96, P less than 0.005) between pH 6.77 and 7.74. The pH effect was completely reversible, did not require either CO2 or HCO3-, and was not affected by the presence of mucosal barium at 1 mM. Mucosal bumetanide (0.1 mM) completely inhibited the pH effect. These data suggest that the process by which Cl- is absorbed in the flounder intestine is sensitive to pH. The data do not indicate whether pH affects Na+-K+-2Cl- cotransport or a Cl- transport pathway in series with this process. The direction of Cl- absorption in response to pH contrasts with inverse relation of pH and Cl- absorption in mammalian small intestine.


1988 ◽  
Vol 137 (1) ◽  
pp. 277-286 ◽  
Author(s):  
D. N. Crawford ◽  
W. R. Harvey

Ba2+ and Ca2+ prevent and reverse the Btk delta-endotoxin inhibition of the short-circuit current in isolated lepidopteran midgut. These findings support the K+ pump-leak steady-state model for midgut K+ homeostasis and the K+ channel mechanism of Bt toxin action. They provide a new tool with which to study the interactions between Bt toxin and midgut cell membranes.


1985 ◽  
Vol 74 (1) ◽  
pp. 137-152
Author(s):  
B.L. Gupta ◽  
J.A. Dow ◽  
T.A. Hall ◽  
W.R. Harvey

An alkaline hydrolysate of Bacillus thuringiensis var kurstaki HD1 (Btk) parasporal crystals was administered at 25 micrograms ml-1 (f.c.) to isolated, short-circuited, midguts of tobacco hornworm (Manduca sexta) larvae. The short-circuit current (s.c.c.), a precise measure of K+ active transport, was inhibited by 78% in 10 min in Btk-treated midguts as compared to controls. The elemental concentrations of K, together with Na, Mg, P, S, Cl and Ca, as well as the water content, were determined by electron probe X-ray microanalysis (EPXMA) in the muscle cells, columnar cells and goblet cells, as well as in the extracellular goblet cavity and the bathing media. The average K concentration in the goblet cell cavity was 129 mmol/kg wet wt in control midguts but only 37 mmol/kg wet wt in Btk-treated midguts. The elemental concentrations, including that of K, in other cell compartments were much less affected by Btk, but a rise in total cell calcium is suggested. It has been previously suggested that in vitro Btk acts specifically on limited regions of the apical membrane of the midgut epithelial cells. The simplest interpretation of the EPXMA results would be that initially Btk interacts specifically with the goblet cell apical membrane, which bounds the goblet cavity and contains the K+ pump responsible for the s.c.c. and high transepithelial potential difference (p.d.). Such interaction results in a rapid disruption of K+ transport across the goblet cell apical membrane, leading to dissipation of the K+ gradient and loss of p.d. The histopathological changes previously reported by other workers would then be a consequence of K+ pump inhibition causing changes in the intracellular pH, Ca2+ etc. Some possible molecular bases for these specific interactions between Btk and cell membrane are discussed.


1983 ◽  
Vol 244 (6) ◽  
pp. F639-F645 ◽  
Author(s):  
M. J. Welsh

Addition of barium ion, Ba2+, to the submucosal bathing solution of canine tracheal epithelium reversibly decreased the short-circuit current and increased transepithelial resistance. The decrease in short-circuit current represented a decrease in the net rate of Cl secretion with no change in the rate of Na absorption. Intracellular microelectrode techniques and an equivalent electrical circuit analysis were used to localize the effect of Ba2+ to an inhibition of the permeability of the basolateral membrane to K. Ba2+ (2 mM) doubled basolateral membrane resistance, decreased the equivalent electromotive force at the basolateral membrane, and decreased the magnitude of the depolarization of basolateral membrane voltage produced by increasing the submucosal K concentration. The inhibition of the basolateral K permeability depolarized the negative intracellular voltage, resulting in both a decrease in the driving force for Cl exit and an estimated increase in intracellular Cl concentration. These studies indicate that there is a Ba2+-inhibitable K conductance at the basolateral membrane of tracheal epithelial cells and that the K permeability plays an important role in the generation of the negative intracellular electrical potential that provides the driving force for Cl exit from the cell.


2000 ◽  
Vol 279 (3) ◽  
pp. R925-R935 ◽  
Author(s):  
Mario Díaz ◽  
Virtudes Medina ◽  
Tomás Gómez ◽  
Antonio Lorenzo

The active Na+-independent transport ofl-alanine across the duodenal mucosa of the lizard Gallotia galloti was studied in Ussing-type chambers using a computer-controlled voltage clamp. Addition of l-alanine to the Na+-free bathing solutions resulted in a significantl-alanine absorption ( J net) that was paralleled by an increase in transepithelial short-circuit current ( I sc) and potential difference (PD) without apparent changes in the tissue conductance. The concentration dependence of J net, PD, and I sc displayed Michaelis-Menten kinetics.l-alanine-induced electrical changes were completely inhibited by external alkaline pH or by the H+-ionophore carbonyl cyanide m-chlorophenyl-hydrazone in the bathing solution. The alanine-induced electrogenicity was dependent on the presence of extracellular K+ and could be blocked by serosal Ba2+ or mucosal orthovanadate. These results suggest the existence of an H+-coupledl-alanine cotransport at the apical membrane of enterocytes. The favorable H+ driving force is likely to be maintained by an apical vanadate-sensitive H+-K+-ATPase, allowing the extrusion of H+ in an exchange with K+. Potassium exit through a basolateral barium-sensitive conductance provides the key step for the electrogenicity of l-alanine absorption.


1986 ◽  
Vol 250 (5) ◽  
pp. F781-F784 ◽  
Author(s):  
S. Tsuboi ◽  
R. Manabe ◽  
S. Iizuka

Transport of Na and Cl across the isolated dog retinal pigment epithelium (RPE) choroid was investigated. Under the short-circuit condition, a net Na flux was observed from choroid to retina and a net Cl flux was determined in the opposite direction. The current created by the net flux of these two ions was larger than the short-circuit current (SCC). Addition of 10(-5) M ouabain to the apical side inhibited net fluxes of both Na and Cl, whereas it reduced the SCC 84%. Addition of 10(-4) M furosemide to the apical side inhibited net Cl flux but had no effect on the net Na transport. The 10(-4) M furosemide reduced the SCC 38%. These drugs had no effect when applied to the basal side. Thus the transport of both Na and Cl depends on the Na-K-ATPase in the apical membrane of the dog RPE. A furosemide-sensitive neutral carrier at the apical membrane is suggested for the transport of Cl. Replacement of HCO3 with SO4 in the bathing solution caused an increase in the SCC, indicating the choroid-to-retina movement of HCO3 across the short-circuited dog RPE choroid.


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