scholarly journals AN INVESTIGATION OF THE MIDGUT K+ PUMP OF THE TOBACCO HORNWORM (MANDUCA SEXTA) USING SPECIFIC INHIBITORS AND AMPHOTERICIN B

1994 ◽  
Vol 188 (1) ◽  
pp. 191-204 ◽  
Author(s):  
K Schirmanns ◽  
W Zeiske
Keyword(s):  
Amphotericin B ◽  
Manduca Sexta ◽  
Short Circuit ◽  
Short Circuit Current ◽  
K Secretion ◽  
High Doses ◽  
Sites Of Action ◽  
Apical Membranes ◽  
Specific Inhibitors

Active K+ secretion in isolated posterior midguts of Manduca sexta was studied by measuring the short-circuit current. One aim of this study was to verify the postulate from biochemical reports that the cooperative apical arrangement of a vacuolar-type H+-ATPase (V-ATPase) and a K+/H+ antiporter drive the short-circuit current. Hence, we tested several specific inhibitors of the V-ATPase on the in vitro midgut preparation. Nitrate, bafilomycin A1, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) and amiloride all reduced the short-circuit current. This suggests that the H+-ATPase is involved in transepithelial K+ secretion. However, even at relatively high doses of these inhibitors, the block of the short-circuit current was not complete. Two other agents, thallium ions (Tl+, at millimolar concentrations) and trimethyltin chloride (TMT, 50 µmol l-1), did abolish the short-circuit current. Apical, but not basal, use of the ionophore amphotericin B largely eliminated the short-circuit current. This supports the view that the current-generating source resides in the apical membranes. An apical (and probably intracellular) site of action for NO3-, Tl+ and TMT is suggested by the observation that basal amphotericin B is needed for blockage by NO3- but does not, however, influence the effect of Tl+ and TMT. Likely sites of action are the V-ATPase (for nitrate and TMT) and the K+/H+ antiporter (for Tl+).

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.

scholarly journals Distinct K+ conductive pathways are required for Cl− and K+ secretion across distal colonic epithelium

2006 ◽  
Vol 291 (4) ◽  
pp. C636-C648 ◽  
Author(s):  
Susan Troutman Halm ◽  
Tianjiang Liao ◽  
Dan R. Halm
Keyword(s):  
Colonic Mucosa ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Rat Colon ◽  
Colonic Epithelium ◽  
High Concentration ◽  
K Secretion ◽  
Synergistic Response ◽  
High Concentrations ◽  
Apical Membranes

Secretion of Cl− and K+ in the colonic epithelium operates through a cellular mechanism requiring K+ channels in the basolateral and apical membranes. Transepithelial current [short-circuit current ( Isc)] and conductance ( Gt) were measured for isolated distal colonic mucosa during secretory activation by epinephrine (Epi) or PGE2 and synergistically by PGE2 and carbachol (PGE2 + CCh). TRAM-34 at 0.5 μM, an inhibitor of KCa3.1 (IK, Kcnn4) K+ channels (H. Wulff, M. J. Miller, W. Hänsel, S. Grissmer, M. D. Cahalan, and K. G. Chandy. Proc Natl Acad Sci USA 97: 8151–8156, 2000), did not alter secretory Isc or Gt in guinea pig or rat colon. The presence of KCa3.1 in the mucosa was confirmed by immunoblot and immunofluorescence detection. At 100 μM, TRAM-34 inhibited Isc and Gt activated by Epi (∼4%), PGE2 (∼30%) and PGE2 + CCh (∼60%). The IC50 of 4.0 μM implicated involvement of K+ channels other than KCa3.1. The secretory responses augmented by the K+ channel opener 1-EBIO were inhibited only at a high concentration of TRAM-34, suggesting further that KCa3.1 was not involved. Sensitivity of the synergistic response (PGE2 + CCh) to a high concentration TRAM-34 supported a requirement for multiple K+ conductive pathways in secretion. Clofilium (100 μM), a quaternary ammonium, inhibited Cl− secretory Isc and Gt activated by PGE2 (∼20%) but not K+ secretion activated by Epi. Thus Cl− secretion activated by physiological secretagogues occurred without apparent activity of KCa3.1 channels but was dependent on other types of K+ channels sensitive to high concentrations of TRAM-34 and/or clofilium.

Permeability of the rabbit colon in vitro

1977 ◽  
Vol 232 (1) ◽  
pp. F5-F9 ◽  
Author(s):  
T. Yorio ◽  
P. J. Bentley
Keyword(s):  
Amphotericin B ◽  
Short Circuit ◽  
Physiological Role ◽  
Short Circuit Current ◽  
Rabbit Colon ◽  
Flux Measurements

The rabbit colon, in vitro as everted-sac or diaphragm preparations, exhibits a transmural PD, as high as 70 mV, a short-circuit current (SCC) of 100 to 150 muA cm-3 and a resistance of 300–500 omega-cm2. It maintains these functions for at least 3 h. The SCC can be abolished by amiloride or increased by amphotericin B. Na, Cl, and K flux measurements showed a net influx (mucosa to serosa) of Na and a net efflux of K. The SCC can be accounted for by the movements of these ions. The SCC in the presence of amphotericin B was nearly equivalent to the net Na flux. Amiloride abolished the net Na transfer but did not ht (Ktrnas X 10(7)) for water was 5,000 cm s-1 while that for urea was 26 cm s-1. The rabbit provides a viable pn and the present observations appear to be consistent with its physiological role.

Aldosterone stimulates K secretion prior to onset of Na absorption in guinea pig distal colon

1994 ◽  
Vol 266 (2) ◽  
pp. C552-C558 ◽  
Author(s):  
D. R. Halm ◽  
S. T. Halm
Keyword(s):  
Guinea Pig ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Ussing Chambers ◽  
High Affinity Receptor ◽  
Additive Increase ◽  
K Secretion ◽  
Affinity Receptor

Distal colon from guinea pig was stimulated in vitro by aldosterone in Ussing chambers that allowed measurement of short-circuit current (Isc) and tissue conductance (Gt). The response to aldosterone was delayed by approximately 20 min and resulted in a negative Isc, consistent with K secretion. Approximately 1 h later the Isc began to increase and eventually became positive, consistent with subsequent stimulation of Na absorption. The Na-absorptive response could be inhibited by mucosal amiloride without altering the rate of K secretion. Similarly, K secretion could be inhibited by serosal bumetanide without altering Na absorption. In the presence of spironolactone, actinomycin D, or cycloheximide, aldosterone failed to stimulate both K secretion and Na absorption. A dose response to aldosterone provided an apparent Kd of 2.6 +/- 0.5 nM, consistent with a high-affinity receptor coupled to this secretory response. Stimulation by the K secretagogue epinephrine did not produce an additive increase in K secretion, suggesting that the same cell type responds to both aldosterone and epinephrine and that the protein induced by aldosterone was not one of the membrane proteins responsible for K secretion.

scholarly journals K+ CHANNEL PERMEATION AND BLOCK IN THE MIDGUT EPITHELIUM OF THE TOBACCO HORNWORM MANDUCA SEXTA

1994 ◽  
Vol 197 (1) ◽  
pp. 179-200
Author(s):  
K Schirmanns ◽  
W Zeiske
Keyword(s):  
Manduca Sexta ◽  
Short Circuit ◽  
Short Circuit Current ◽  
K Channel ◽  
K Channels ◽  
Zero Current ◽  
Channel Opening ◽  
Voltage Dependent ◽  
K Current

The K+-secreting larval midgut of Manduca sexta in vitro was voltage- or current-clamped. In contrast to Tl+, NH4+ and Na+, both Rb+ and K+ generated a short-circuit current, although with different saturation kinetics. The dependence of the short-circuit current on Rb+/K+ mole fraction gave no evidence for multi-ion occupation of the basolateral K+ channels. After 'functionally' eliminating the apical membranes using the ionophore amphotericin B and the 'apical K+ pump' blockers trimethyltin chloride or Tl+, the K+ channels could be more closely investigated. By measuring zero-current potentials, permeability ratios PX/PK were estimated using an adapted version of the Goldman­Hodgkin­Katz voltage equation. Their sequence was K+ (1) = Tl+ > Rb+ (0.38) > NH4+ (~0.3) > Cs+ (0.03) > Na+ (~0). The K+ channels could not be blocked by basally applied Cs+, Na+ or tetraethylammonium. Blockade of K+ current by Ba2+ was typically voltage-dependent, but only at moderate transbasal voltages. The relative electrical distance delta of the Ba2+ binding site from the basal channel opening was determined to be 0.2. At zero transbasal voltage, the apparent inhibition constant for barium KBa* was 1.7 mmol l-1.

Potassium secretion by rabbit descending colon: effects of adrenergic stimuli

1986 ◽  
Vol 250 (4) ◽  
pp. G432-G439 ◽  
Author(s):  
P. L. Smith ◽  
R. D. McCabe
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Maximal Effect ◽  
Dependent Manner ◽  
Adrenergic Stimulation ◽  
Adrenergic Agents ◽  
Ussing Chambers ◽  
K Secretion ◽  
Potassium Secretion

Stripped rabbit distal colonic mucosa was studied in vitro in Ussing chambers to investigate the effects of adrenergic stimuli on Na+, K+, and Cl- transport. The adrenergic stimuli epinephrine and norepinephrine decrease short-circuit current in a dose-dependent manner, with a half-maximal effect at 5 X 10(-7) M and a maximal effect between 10(-5) and 10(-4) M. The effects produced by norepinephrine and epinephrine can also be elicited by the beta 1-agonist dobutamine, but not by the beta 2-agonist terbutaline or the alpha-agonist phenylephrine. In addition, the effects of adrenergic stimulation can be inhibited by the beta-antagonist propranolol but not by the muscarinic antagonist atropine, the alpha 2-antagonist yohimbine, or tetrodotoxin. The decrease in short-circuit current elicited by adrenergic stimuli is accompanied by an increase in net K+ secretion with no change in net Cl- or Na+ transport. This increase in net K+ secretion elicited by beta-adrenergic stimulation can be inhibited by trifluoperazine but not by indomethacin. These studies suggest that K+ transport by the colon can be regulated by adrenergic agents acting via beta 1-receptors.

Potassium secretion by nonsensory region of gerbil utricle in vitro

1987 ◽  
Vol 253 (4) ◽  
pp. F613-F621 ◽  
Author(s):  
N. Y. Marcus ◽  
D. C. Marcus
Keyword(s):  
Short Circuit ◽  
Control Level ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Electrical Potential Difference ◽  
Free Solution ◽  
K Secretion

The isolated nonsensory region of the gerbil utricle in vitro produced a lumen-positive transepithelial electrical potential difference (VT) of +5.7 mV and a luminal fluid containing 106 mM K when bathed in mammalian Ringer solution (5 mM K and 150 mM Na). The lumen of this region was perfused in vitro with K-free solution and the luminal [K], VT, and transepithelial resistance (RT) were measured before and following perfusion under control conditions and after addition of bumetanide (0.1 mM) or ouabain (1 mM) to the bath. The perfusate contained a reduced [Ca], since the average value of utricular endolymph in vivo (0.28 +/- 0.03 mM) measured with Ca-selective microelectrodes was 38% of that in perilymph. Under control conditions, the luminal [K] initially increased at a rate of 2.13 mumol X cm-2 X h-1 after perfusion; net secretion continued until the luminal [K] returned to its preperfusion level. This flux rate corresponds to 57 microA/cm2. The “equivalent short-circuit current” (Equiv. Isc; VT/RT) was found to average 61 microA/cm2. Both K secretion and VT were fully inhibited by bumetanide and by ouabain. Luminal application of Ba (5 mM) in K-free solution had no effect on the initial rate of K secretion, but did prevent full recovery of luminal [K] to the control level. These results are the first estimates of K secretion by the nonsensory cells of the utricle and are the first to directly demonstrate inhibition of K secretion in the inner ear by bumetanide and in the nonsensory tissue of the utricle by ouabain.

Comparison of effects of standard diuretics and indanone in isolated toad cornea and bladder

1980 ◽  
Vol 238 (1) ◽  
pp. R70-R75
Author(s):  
J. R. Schnieders ◽  
J. H. Ludens
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Suitable Model ◽  
Thick Ascending Limb ◽  
Loop Of Henle ◽  
Site Of Action ◽  
Diuretic Agents ◽  
Sites Of Action

Short-circuit current (SCC) techniques were used to monitor effects of various diuretic agents on Na+ transport in toad bladder and Cl- transport in toad cornea. In bladder, various agents from different "classes" of diuretics inhibited SCC whereas in cornea only "loop diuretics," i.e., those with a primary site of action in the ascending limb of the loop of Henle, inhibited SCC. Classification of the diuretics based on sensitivity in cornea and bladder revealed that diuretics with a common renal site of action gravitated into common classification groups. Thus, our studies suggested that cornea may be a suitable model of the thick ascending limb of the loop of Henle and that cornea and bladder, studied jointly, may serve as an in vitro system for predicting potential renal sites of action of new diuretics. This system, when used to characterize the new diuretic indanone, revealed that this agent, in all respects, displayed the characteristics of a loop diuretic.

Effects of amphotericin B on ionic transport and sodium permeability of the toad lens

1975 ◽  
Vol 229 (6) ◽  
pp. 1520-1525 ◽  
Author(s):  
PJ Bentley ◽  
OA Candia
Keyword(s):  
Amphotericin B ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Anterior Surface ◽  
Sodium Permeability ◽  
Solution Bathing ◽  
Net Flux ◽  
Large Decline

The polyene antibiotic amphotericin B decreases the PD and short-circuit current (SCC) across the amphibian lens in vitro. It was only effective when placed in the solution at the anterior side and its effect was reversible. Amphotericin B caused a large decline in the PD across the anterior surface of the lens and a smaller reduction in the PD across the posterior side. This seems to be due to a direct decrease of the electrical resistance of the anterior face. The effects required the presence of sodium in the Ringer solution bathing the anterior surface. The translenticular Na fluxes were increased in both directions so that the net flux changed little. Amphotericin B produced a considerable increase in the rate of accumulation of sodium and loss of potassium by the lens. The oxygen consumption of the lens was unchanged by amphotericin B. Amphotericin B appears to act on the lens epithelium by selectively increasing its passive sodium permeability.

Ion transport, ciliary activity, and mechanosensitivity of sinusal mucosa: an in vitro study

1996 ◽  
Vol 271 (3) ◽  
pp. L349-L358 ◽  
Author(s):  
D. Leuba ◽  
Y. De Ribaupierre ◽  
P. Kucera
Keyword(s):  
Fluid Transport ◽  
Short Circuit ◽  
Fluid Velocity ◽  
In Vitro Study ◽  
Short Circuit Current ◽  
Transepithelial Transport ◽  
Ciliary Activity ◽  
Apical Side ◽  
K Secretion

The hypothesis that relative movement between respiratory epithelium and surrounding fluid modulates both ciliary activity and mucosal secretion-absorption properties was tested. Fresh human (HM) and bovine sinusal mucosae (BM) were mounted in transparent chambers perfused with defined media at 36 degrees C. The fluid advanced along the ciliated surface at 15-30 microns/s. Transepithelial transport of electrolytes was studied by using voltage-clamp technique. Ciliary beating frequency (CBF) was recorded by using laser reflectometry. Average transmucosal potential difference (apical side negative) and short-circuit current (Isc) were -0.5 mV and 17 microA.cm-2 for HM and -1.4 mV and 28 microA.cm-2 for BM. Average CBF was 900 beats/min. Ionic pathways included basolateral Na,K-ATPase, K+ channels and symports for Na-Cl and Na-glucose, and apical channels for Na+ (absorption) and for Cl- and K+ (secretion). Increase of fluid velocity up to 300 microns/s induced significant increase of both Isc (63%) and CBF (29%). Such adaptations of transepithelial fluid transport and ciliary activity to hydrodynamic conditions might reflect a mechanism of coordination between the secretion of mucus electrolytes and mucociliary clearance.

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