Comparison of Potassium Transport in Three Structurally Distinct Regions of The Insect Midgut

1981 ◽  
Vol 91 (1) ◽  
pp. 103-116
Author(s):  
MOIRA CIOFFI ◽  
WILLIAM R. HARVEY
Keyword(s):  
Goblet Cell ◽  
Apical Membrane ◽  
Close Association ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Potassium Transport ◽  
Structural Requirement ◽  
Posterior Midgut ◽  
New Information ◽  
Structural And Functional Properties

1. Active potassium transport across the isolated midgut of the Tobacco Hornworm larva, Manduca sexta, was studied by measuring the short circuit current (ISC) and unidirectional 42-potassium fluxes. 2. The midgut is composed of structurally distinct anterior, middle and posterior regions, all of which are shown to transport potassium, so that by comparing and contrasting their structural and functional properties new information on the mechanism of midgut potassium transport was obtained. 3. It has previously been shown that the potassium pump is located on the apical membrane of the goblet cell. In the anterior and middle regions of the midgut the goblet cell has a large cavity and mitochondria are closely associated with the apical membrane while in the posterior midgut the goblet cavity is much smaller, and mitochondria are not associated with the apical membrane. However, the apical membrane particles which have been implicated in active potassium transport in a number of other insect epithelia are present in all three regions. This observation suggests that the particles are a structural requirement for active transport, and that close association between mitochondria and the transporting membrane is not essential. 4. Comparison of the kinetic influx pool size and the differences in the ISC decay profiles between the three midgut regions suggest that part of the influx pool is a transported pool located in the goblet cavity. 5. A new model to explain the driving force for potassium transport in the midgut is proposed, in which the rate of potassium transport controls the entrance of potassium into the cell, rather than the opposite, currently accepted view.

Electron probe X-ray microanalysis of the effects of Bacillus thuringiensis var kurstaki crystal protein insecticide on ions in an electrogenic K+-transporting epithelium of the larval midgut in the lepidopteran, Manduca sexta, in vitro

1985 ◽  
Vol 74 (1) ◽  
pp. 137-152
Author(s):  
B.L. Gupta ◽  
J.A. Dow ◽  
T.A. Hall ◽  
W.R. Harvey
Keyword(s):  
Bacillus Thuringiensis ◽  
Manduca Sexta ◽  
Goblet Cell ◽  
Electron Probe ◽  
Apical Membrane ◽  
Short Circuit ◽  
Short Circuit Current ◽  
X Ray ◽  
Elemental Concentrations

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.

Active transport of magnesium across the isolated midgut of Hyalophora cecropia

1975 ◽  
Vol 63 (2) ◽  
pp. 313-320
Author(s):  
J. L. Wood ◽  
A. M. Jungreis ◽  
W. R. Harvey
Keyword(s):  
Steady State ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Potassium Transport ◽  
Magnesium Concentration ◽  
Magnesium Transport ◽  
Wet Weight ◽  
Net Flux ◽  
Burk Plot

1. The 28Mg-measured net flux of magnesium from lumen-side to haemolymph-side of the isolated and short-circuited midgut was 1.97 +/− 0.28 mu-equiv cm(−2) /(−1) in 8 mM-Mg2+. 2. The magnesium-influx shows a delay before the tracer steady-state is attained, indicating the existence of a magnesium-transport pool equivalent to 6.7 mu-equiv/g wet weight of midgut tissue. 3. Magnesium depresses the short-circuit current produced the midgut but not the potassium transport, the depression being equal to the rate of magnesium transport. 4. Magnesium transport yields a linear Lineweaver-Burk plot with an apparent Km of 34 mM-Mg2+ and an apparent Vmax of 14.9 mu-equiv cm(−1) /(−1). 5. Magnesium is actively transported across the midgut and contributes to the regulation of the haemolymph magnesium concentration in vivo.

Bathing Solution Tonicity and Potassium Transport by the Midgut of the Tobacco Hornworm Manduca Sexta

1979 ◽  
Vol 78 (1) ◽  
pp. 213-223
Author(s):  
DAVID F. MOFFETT
Keyword(s):  
Manduca Sexta ◽  
Short Circuit ◽  
Weight Ratio ◽  
Short Circuit Current ◽  
Dry Weight ◽  
Potassium Transport ◽  
Bathing Solution ◽  
Histological Studies ◽  
Wet Weight ◽  
Small Solute

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.

An electrogenic amino acid transporter in the apical membrane of cultured human bronchial epithelial cells

1998 ◽  
Vol 275 (5) ◽  
pp. L917-L923 ◽  
Author(s):  
Luis J. V. Galietta ◽  
Luciana Musante ◽  
Leila Romio ◽  
Ubaldo Caruso ◽  
Annarita Fantasia ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Amino Acid ◽  
Epithelial Cells ◽  
Apical Membrane ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Bronchial Epithelial Cells ◽  
Maximal Effect ◽  
Human Bronchial Epithelial Cells ◽  
Bronchial Epithelial

We performed Ussing chamber experiments on cultured human bronchial epithelial cells to look for the presence of electrogenic dibasic amino acid transport. Apical but not basolaterall-arginine (10–1,000 μM) increased the short-circuit current. Maximal effect and EC50were ∼3.5 μA/cm2and 80 μM, respectively, in cells from normal subjects and cystic fibrosis patients. The involvement of nitric oxide was ruled out because a nitric oxide synthase inhibitor ( NG-nitro-l-arginine methyl ester) did not decrease the arginine-dependent current. Apicall-lysine,l-alanine, andl-proline, but not aspartic acid, were also effective in increasing the short-circuit current, with EC50values ranging from 26 to 971 μM. Experiments performed with radiolabeled arginine demonstrated the presence of an Na+-dependent concentrative transporter on the apical membrane of bronchial cells. This transporter could be important in vivo to maintain a low amino acid concentration in the fluid covering the airway surface.

Role of alpha 1- and alpha 2-adrenergic receptors in Cl- transport across frog corneal epithelium

1988 ◽  
Vol 255 (6) ◽  
pp. C724-C730 ◽  
Author(s):  
T. C. Chu ◽  
O. A. Candia
Keyword(s):  
Adenylate Cyclase ◽  
Corneal Epithelium ◽  
Apical Membrane ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Negative Influence ◽  
Adenylate Cyclase System ◽  
Positive Effects ◽  
Cl Transport ◽  
Beta Agonists

Norepinephrine, 10(-6) M, reduced Cl- transport by 26% in 75% of isolated frog corneal epithelia. This inhibition was not previously reported. Since beta-adrenergic agonists are known to only stimulate Cl- transport, the action of specific alpha 1- and alpha 2-agonists on Cl- transport and electrical parameters was investigated. Phenylephrine, an alpha 1-agonist always stimulated the Cl(-)-dependent short-circuit current (Isc), but less than the beta-agonists. UK-14,304-18 (UK), a selective alpha 2-agonist, reduced both the Isc (by 31% at 10(-5) M) and the stroma-to-tear unidirectional Cl- flux. UK hyperpolarized the apical membrane potential difference and increased the transepithelial resistance and apical-to-basolateral resistance ratio. UK reduced forskolin-stimulated adenylate cyclase activity by 36%. The electrophysiological effects of UK are consistent with a reduction of the Cl- permeability at the apical membrane. Pretreatment with UK sensitized the tissue for a greater effect by forskolin. Results show that the frog corneal epithelium also possesses alpha 1- and alpha 2-receptors, the latter negatively coupled to the adenylate cyclase system. Cl- transport is thus regulated by an interaction between the positive effects of beta- and alpha 1-stimulation and the negative influence of alpha 2-stimulation.

Active Transport by the Cecropia Midgut

1968 ◽  
Vol 48 (1) ◽  
pp. 1-12
Author(s):  
W. R. HARVEY ◽  
J. A. HASKELL ◽  
S. NEDERGAARD
Keyword(s):  
Transport Mechanism ◽  
Potassium Concentration ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Potassium Transport ◽  
Nernst Equation ◽  
Tissue Concentrations ◽  
Sodium Magnesium ◽  
Lines Of Evidence ◽  
Stimulation Of

1. From two lines of evidence, we conclude that the potassium transport gives rise directly to the midgut potential, i.e. that the active potassium transport mechanism is electrogenic. 2. First, diffusion potentials of neither potassium, sodium, magnesium, calcium, nor chloride could give rise to the large midgut potential if values for tissue concentrations are accepted for their respective activities in the epithelium. 3. Secondly, no externally added cation other than potassium is required to sustain either the potential or short circuit current, no specific anion is required, and no metabolic ion is known to be produced in sufficient amount to act as a counter ion for potassium in a non-electrogenic process. 4. Changes in the concentration of potassium on the blood-side of the midgut always lead to changes in potential in the direction predicted by the Nernst equation. Moreover, a tenfold change in potassium concentration leads to the expected 59 mV. potential change provided that the prior midgut potential is at least 130 mV. This effect could be attributed either to the stimulation of an electrogenic potassium pump or to a potassium diffusion potential across the blood-side barrier.

ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia

2001 ◽  
Vol 281 (2) ◽  
pp. C633-C648 ◽  
Author(s):  
Sasha Blaug ◽  
Kevin Hybiske ◽  
Jonathan Cohn ◽  
Gary L. Firestone ◽  
Terry E. Machen ◽  
...  
Keyword(s):  
Tight Junctions ◽  
Fluid Transport ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Equivalent Circuit Analysis ◽  
Mean Values ◽  
Apical Side

Mammary epithelial 31EG4 cells (MEC) were grown as monolayers on filters to analyze the apical membrane mechanisms that help mediate ion and fluid transport across the epithelium. RT-PCR showed the presence of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial Na+ channel (ENaC) message, and immunomicroscopy showed apical membrane staining for both proteins. CFTR was also localized to the apical membrane of native human mammary duct epithelium. In control conditions, mean values of transepithelial potential (apical-side negative) and resistance ( R T) are −5.9 mV and 829 Ω · cm2, respectively. The apical membrane potential ( V A) is −40.7 mV, and the mean ratio of apical to basolateral membrane resistance ( R A/ R B) is 2.8. Apical amiloride hyperpolarized V A by 19.7 mV and tripled R A/ R B. A cAMP-elevating cocktail depolarized V A by 17.6 mV, decreased R A/ R B by 60%, increased short-circuit current by 6 μA/cm2, decreased R T by 155 Ω · cm2, and largely eliminated responses to amiloride. Whole cell patch-clamp measurements demonstrated amiloride-inhibited Na+ currents [linear current-voltage ( I-V) relation] and forskolin-stimulated Cl−currents (linear I-V relation). A capacitance probe method showed that in the control state, MEC monolayers either absorbed or secreted fluid (2–4 μl · cm−2 · h−1). Fluid secretion was stimulated either by activating CFTR (cAMP) or blocking ENaC (amiloride). These data plus equivalent circuit analysis showed that 1) fluid absorption across MEC is mediated by Na+ transport via apical membrane ENaC, and fluid secretion is mediated, in part, by Cl− transport via apical CFTR; 2) in both cases, appropriate counterions move through tight junctions to maintain electroneutrality; and 3) interactions among CFTR, ENaC, and tight junctions allow MEC to either absorb or secrete fluid and, in situ, may help control luminal [Na+] and [Cl−].

Forskolin-induced HCO3- current across apical membrane of the frog corneal epithelium

1990 ◽  
Vol 259 (2) ◽  
pp. C215-C223 ◽  
Author(s):  
O. A. Candia
Keyword(s):  
Corneal Epithelium ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Pump Current ◽  
Gas Composition ◽  
Apical Side ◽  
Disulfonic Stilbene ◽  
Stimulation Of

Forskolin (and other Cl- secretagogues) does not affect the very small Na(+)-originated short-circuit current (Isc) across frog corneal epithelium bathed in Cl- free solutions. However, forskolin in combination with increased PCO2 bubbling of the solutions (5-20% CO2) stimulated Isc proportionally to PCO2 to a maximum of approximately 8 microA/cm2. This current could be eliminated and reinstated by sequentially changing the gas composition of the bubbling to 100% air and 20% CO2-80% air. The same effects were observed when PCO2 changes were limited to the apical-side solution. Stroma-to-tear HCO3- movement was deemed unlikely, since the increase in Isc was observed with a HCO3(-)-free solution on the stromal side and CO2 gassing limited to the tear side. From the effects of ouabain and tryptamine, at least 80% of the Isc across the basolateral membrane can be accounted for by the Na+ pump current plus K+ movement from cell to bath. Methazolamide also inhibited Isc. Current across the apical membrane cannot be attributed to an electronegative Na(+)-HCO3- symport given the insensitivity of Isc to a disulfonic stilbene and the fact that stroma-to-tear Na+ fluxes did not increase on stimulation of Isc. The tear-to-stroma Na+ flux also remained unaltered, negating an increased apical bath-to-cell Na+ flow. The forskolin-20% CO2 manipulation produced a depolarization of the intracellular potential, a reduction in the apical-to-basolateral resistance ratio, and a decrease in transepithelial resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Apical nonspecific cation conductances in rabbit cecum

1994 ◽  
Vol 266 (3) ◽  
pp. G475-G484 ◽  
Author(s):  
J. H. Sellin ◽  
W. P. Dubinsky
Keyword(s):  
Epithelial Transport ◽  
Apical Membrane ◽  
Divalent Cations ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Na Channel ◽  
Similar Response ◽  
Electrogenic Transport

Rabbit cecum exhibits electrogenic Na absorption in vitro. However, because this transport process is not inhibited by amiloride nor does it demonstrate saturation kinetics typical of the amiloride-inhibitable Na channel, we considered whether the cecal transporter represented one of a recently described family of nonselective cation conductances or channels (NSCC). Both transepithelial and vesicle studies demonstrated that K, Cs, and Rb were transported via an apical conductance. Electrogenic transport was inhibited by divalent cations including Ca, Mg, and Ba but was unaffected by either lanthanum or gadolinium. Parallel studies in distal colon did not exhibit a similar response to either K substitution or Ba inhibition. Phenamil, verapamil, and nicardipine significantly inhibited the short-circuit current (Isc). stimulated by nominal Ca- and Mg-free conditions. Flux studies demonstrated a correlation between changes in Isc and Na transport. Microelectrode impalement studies suggested that there may be both NSCC and K conductance in the apical membrane. Planar bilayer studies identified a 190-pS cation channel that may correlate with the macroscopic transport properties of this epithelium. These studies are consistent with a model of cecal Na absorption mediated by a NSCC in the apical membrane; this may be the mechanism underlying the distinct epithelial transport characteristics of this intestinal segment.

Role of CFTR in chloride secretion across human tracheal epithelium

1995 ◽  
Vol 269 (5) ◽  
pp. L561-L566 ◽  
Author(s):  
B. Q. Shen ◽  
R. J. Mrsny ◽  
W. E. Finkbeiner ◽  
J. H. Widdicombe
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Culture Conditions ◽  
Tracheal Epithelium ◽  
Disulfonic Acid ◽  
Acetoxymethyl Ester ◽  
Cl Channels

We have tested two hypotheses: 1) the cystic fibrosis transmembrane conductance regulator (CFTR) represents the predominant Cl conductance in the apical membrane of human tracheal epithelium, and 2) CFTR in this tissue is close to maximally activated under baseline conditions. In support of the first hypothesis, we found 1) when the level of differentiation of cultures was varied by varying the culture conditions, there was a significant positive correlation between the levels of CFTR and the magnitude of mediator-induced Cl secretion. 2) Amiloride-insensitive baseline short-circuit current (Isc) and mediator-induced increases in Isc were inhibited by diphenylamine-2-carboxylic acid (DPAC) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a pharmacology consistent with passage of apical membrane Cl current through CFTR; Ca-activated Cl channels are inhibited by DIDS but not by DPAC. 3) Raising temperature from 22 degrees to 37 degrees C increased 125I efflux, and this increase was inhibited by DPAC and blockers of protein kinase A, but not by DIDS or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester. In support of the second hypothesis, we have earlier shown [M. Yamaya, W.E. Finkbeiner, S.Y. Chun, and J.H. Widdicombe. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L713-L724, 1992] that adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents are essentially without effect on Isc across primary cultures of human tracheal epithelium. Here, we further show that these agents are also usually without effect on 125I efflux; the mean increase in efflux in response to elevating cAMP was approximately 20% that of raising temperature from 22 degrees to 37 degrees C.

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