Purification and characteristics of the chloride transport stimulating factor from locust corpora cardiaca: a new peptide

1980 ◽  
Vol 58 (10) ◽  
pp. 1851-1860 ◽  
Author(s):  
J. E. Phillips ◽  
W. Mordue ◽  
J. Meredith ◽  
J. Spring
Keyword(s):  
Chloride Transport ◽  
Short Circuit ◽  
Gel Filtration ◽  
Short Circuit Current ◽  
Water Soluble ◽  
Cellulose Acetate Electrophoresis ◽  
Corpora Cardiaca ◽  
Ph Range ◽  
And Storage ◽  
Layer Chromatography

Corpora cardiaca (CC), cAMP, and hemolymph all increase short-circuit current (Isc) and electropotential difference (PD) across locust rectum by stimulating electrogenic transport of Cl− from the lumen. Using ΔIsc as a bioassay, we have purified the water-soluble stimulant (CTSH) from CC using gel filtration chromatography, DEAE-Sephadex anion exchange, cellulose acetate electrophoresis, and thin-layer chromatography. A single peak of CTSH activity was observed after all these procedures, although small amounts of CTSH activity occasionally remained in the high molecular weight (MW) protein precipitate. CTSH was purified more than 100-fold on Bio-Gel P-30 columns. It has a MW of 8 000 – 12 000, is destroyed by trypsin digestion, and has a net negative charge over the pH range (5–10) at which it is most stable. Various properties (i.e., stability at 20 °C, localization in CC, MW, Rf values) and reciprocal bioassay s indicate that CTSH is different from diuretic, antidiuretic, and adipokinetic hormones from locust CC. No difference in the properties of CTSH from glandular (GL) and storage lobes (SL) of CC were noted, although 80% of activity was in the SL. The concentration of purified CTSH required to cause maximal stimulation of rectal Isc is less than 7 nM.

Pectic enzymes produced by Pseudomonas fluorescens, an organism associated with “pink eye” disease of potato tubers

1972 ◽  
Vol 50 (12) ◽  
pp. 2479-2488 ◽  
Author(s):  
S. S. Hagar ◽  
G. A. McIntyre
Keyword(s):  
Pseudomonas Fluorescens ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Pectin Methylesterase ◽  
Viscosity Reduction ◽  
Cellulose Acetate Electrophoresis ◽  
Pectic Enzymes ◽  
Ph Range ◽  
Two Peaks ◽  
Layer Chromatography

No pectin methylesterase (PME) activity was observed in crude or dialyzed extracts from macerated potato tuber tissue inoculated with Pseudomonas fluorescens; however, pectic lyase (syn. polygalacturonic acid transeliminase, PATE) activity was observed. Two PATE enzymes (peaks 1 and 2) were eluted from a pH 9.4 DEAE-cellulose column using a gradient of pH 7.6 Tris-HCl buffer (0.01–0.1 M). Enzyme in peak 1 was about 6 times more active than enzyme in peak 2 based on reducing group assays, and 10 times more active in viscosity reduction of 1% Na-polypectate (NaPP) at pH 8.5. No increase in absorbancy was observed at 515 nm of clarified reaction mixtures, indicating that saturated oligouronides did not accumulate. Other properties of the two peaks: optimum pH range was 8.5–9.5, substrate preference was NaPP vs. pectin, addition of Ca2+ (0.001 M) enhanced activity while EDTA (0.001 M) decreased activity to [Formula: see text], cellulose acetate electrophoresis revealed one band of protein per peak, and heat of inactivation was 51–60C. Thin-layer chromatography of hydrolytic products from NaPP revealed unsaturated uronides and pectic fragments after 2 h hydrolysis; after 96 h hydrolysis only unsaturated uronides were observed. Molecular weight estimations by Sephadex G-200 gel filtration were about 18 000 (peak 1) and 22 500 (peak 2). Enzyme in either peak macerated 400-μ sections of potato tissue.

Na and Cl Transport across the Isolated Anterior Intestine of the Plaice Pleuronectes Platessa

1981 ◽  
Vol 90 (1) ◽  
pp. 123-142
Author(s):  
M. M. P. RAMOS ◽  
J. C. ELLORY
Keyword(s):  
Chloride Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Negative Potential ◽  
Loop Diuretics ◽  
Significant Part ◽  
Cl Transport ◽  
Anterior Intestine ◽  
Short Circuit Currents ◽  
Na Uptake

1. The tissue was found to have a serosa negative potential, and short-circuit currents equivalent to the net Cl transport. 2. A significant part of the Cl uptake was Na dependent and a similar fraction of the Na uptake was Cl dependent. 3. Short-circuit current and uptake of both ions were inhibited by loop diuretics and analogues. 4. I80 and P.D. were abolished by ouabain. 5. The observations are consistent with the idea of a coupled NaCl entry into the cell, using the energy inherent in the Na gradient; Na being pumped out of the cells by the Na pump and followed electrically by Cl−. Net chloride transport and the serosa negative potential would be a consequence of the permselective properties of the junctions allowing Na but not Cl to recycle back to the mucosal solution.

scholarly journals Further Observations on the Regulation of KCl ABSORPTION ACROSS LOCUST RECTUM

1985 ◽  
Vol 116 (1) ◽  
pp. 153-167
Author(s):  
J. W. HANRAHAN ◽  
J. E. PHILLIPS
Keyword(s):  
Chloride Transport ◽  
Short Circuit ◽  
Membrane Conductance ◽  
Short Circuit Current ◽  
Membrane Resistance ◽  
Open Circuit ◽  
High K ◽  
K Concentration ◽  
K Permeability

1. Electrophysiological and tracer flux techniques were used to studyregulation of KC1 reabsorption across locust recta. Physiologically high K+levels (100 mmolI−1) on the lumen side stimulated net 36Cl flux and reduced the theoretical energy cost of anion transport under open-circuit conductions. 2. The stimulation of short-circuit current (Ibc i.e. active C− absorption) by crude corpora cardiaca extracts (CC) was not dependent on exogenous Ca2+. Stimulations of Ibc were greatly enhanced in the presence of theophylline, indicating that the rate of synthesis of cAMP is increased by CC extracts. High CC levels lowered transepithelial resistance (Rt), suggesting that chloride transport stimulating hormone (CTSH) regulates both active Cl− absorption and counter-ion (K+) permeability. 3. High mucosal osmolarity or K+ concentration decreased Ibc and caused a disproportionately large increase in Rt, consistent with a decrease in theshunt (K+) conductance. Measurements of relative mucosal-to-serosal membrane resistance confirmed that high mucosal K+ levels reduced apical membrane conductance. Lowering mucosal pH to values observed in vivo atthe end of resorptive cycles also inhibited Ibc, apparently without affecting K+ permeability.

Mechanisms of sodium and chloride transport across equine tracheal epithelium

1990 ◽  
Vol 259 (6) ◽  
pp. L459-L467 ◽  
Author(s):  
G. J. Tessier ◽  
T. R. Traynor ◽  
M. S. Kannan ◽  
S. M. O3'Grady
Keyword(s):  
Chloride Transport ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Tracheal Epithelium ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Cotransport System ◽  
Ethanesulfonic Acid

Equine tracheal epithelium, stripped of serosal muscle, mounted in Ussing chambers, and bathed in plasmalike Ringer solution generates a serosa-positive transepithelial potential of 10–22 mV and a short-circuit current (Isc) of 70–200 microA/cm2. Mucosal amiloride (10 microM) causes a 40–60% decrease in Isc and inhibits the net transepithelial Na flux by 95%. Substitution of Cl with gluconate resulted in a 30% decrease in basal Isc. Bicarbonate substitution with 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid decreased the Isc by 21%. The Cl-dependent Isc was inhibited by serosal addition of 1 mM amiloride. Bicarbonate replacement or serosal amiloride (1 mM) inhibits the net Cl flux by 72 and 69%, respectively. Bicarbonate replacement significantly reduces the effects of serosal amiloride (1 mM) on Isc, indicating its effect is HCO3 dependent. Addition of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; 100 microM) causes a 40% increase in Isc. This effect is inhibited by subsequent addition of 10 microM serosal bumetanide. Bumetanide (10 microM) reduces net Cl secretion following stimulation with 8-BrcAMP (100 microM). Serosal addition of BaCl2 (1 mM) causes a reduction in Isc equal to that following Cl replacement in the presence or absence of 100 microM cAMP. These results suggest that 1) Na absorption depends on amiloride-inhibitable Na channels in the apical membrane, 2) Cl influx across the basolateral membrane occurs by both a Na-H/Cl-HCO3 parallel exchange mechanism under basal conditions and by a bumetanide-sensitive Na-(K?)-Cl cotransport system under cAMP-stimulated conditions, and 3) basal and cAMP-stimulated Cl secretion depends on Ba-sensitive K channels in the basolateral membrane.

Active and passive chloride movements across isolated amphibian skin

1964 ◽  
Vol 207 (5) ◽  
pp. 1010-1014 ◽  
Author(s):  
José A. Zadunaisky ◽  
Felisa W. De Fisch
Keyword(s):  
Antidiuretic Hormone ◽  
Chloride Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Ions ◽  
Amphibian Skin ◽  
Chloride Flux ◽  
Bufo Arenarum ◽  
Sodium Loss ◽  
Toad Bufo

Several aspects of chloride passage through isolated amphibian skin were studied. The chloride transport performed by the skin of the frog Leptodactylus ocellatus or the passive chloride fluxes observed in the skin of the toad Bufo arenarum Hensel are not affected by antidiuretic hormone. The chloride transport produces a negative potential and a short-circuit current in sodium-free solutions, though the unidirectional fluxes of chloride are greatly reduced under these conditions. The short-circuit current due to the chloride transport is smaller than the net chloride flux. It was found that this disagreement could be ascribed to a loss of sodium toward the inside from the sodium pool of the skin. Antidiuretic hormone did not affect the chloride current, nor the sodium loss from the skin. The isolated skin of the toad Bufo arenarum Hensel does not transport chloride ions. Thus the active transport of chloride observed in isolated skins of the frog Leptodactylus ocellatus does not depend on environmental conditions, since both animals live in the same surroundings.

Chymotrypsin, ileal chloride transport, and neurotransmitters

1984 ◽  
Vol 247 (3) ◽  
pp. G253-G260 ◽  
Author(s):  
K. A. Hubel
Keyword(s):  
Cell Function ◽  
Chloride Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Electrical Field Stimulation ◽  
Chloride Secretion ◽  
Target Cells ◽  
Flux Chamber ◽  
Rabbit Ileum ◽  
Electrical Field

Electrical field stimulation (EFS) depolarizes nerves and causes chloride secretion by mucosa of rabbit ileum mounted in a flux chamber. To test the hypothesis that the transmitter is a peptide, we determined whether the EFS response was prevented by the endopeptidase chymotrypsin (CT). Serosal, but not mucosal, addition of CT (200 micrograms/ml) reduced the short-circuit current (Isc) response to EFS by 90% or more. CT also reduced Cl absorption by decreasing the mucosal-to-serosal flux, but it did not affect net Na absorption. CT prevented the response to vasoactive intestinal polypeptides, but the response returned when CT activity was eliminated. The response to EFS did not return, however, implying that CT damaged cells that released transmitter or epithelial target cells. CT reduced the Isc response to serotonin by 69% and to A23187 by 10% and did not affect the theophylline response. We conclude that 1) the effects of CT on cell function limit its usefulness in identifying peptide neurotransmitters in epithelium, 2) CT irreversibly inhibits ion transport responses to EFS and to serotonin, and 3) CT reduces absorption of Cl probably by affecting a calcium pathway that modifies Cl transport.

Selective effects of bumetanide on chloride transport in bullfrog cornea

1978 ◽  
Vol 234 (4) ◽  
pp. F297-F301
Author(s):  
O. A. Candia ◽  
H. F. Schoen
Keyword(s):  
Electrical Resistance ◽  
Loop Diuretic ◽  
Chloride Transport ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Selective Inhibitor ◽  
O2 Consumption ◽  
Na Transport ◽  
Bathing Medium

Frog corneas were mounted in a modified Ussing chamber and short-circuit current (SCC) and unidirectional Cl fluxes were measured. Bumetanide, a loop diuretic, at concentrations as low as 10(-7) M, reduced the SCC 29%. At 10(-5) M, bumetanide reduced the SCC 96% and increased transcorneal electrical resistance 20-51%. The forward Cl flux declined from 0.71 +/- 0.04 to 0.20 +/- 0.03 mueq/h.cm2 (n, 7), while, in separate experiments, the backward Cl flux did not change significantly (from 0.22 +/- 0.03 to 0.23 +/- 0.04; n, 7). When corneas were mounted in Cl-free Ringer and the net Na transport was stimulated with amphotericin B, 10(-5) M bumetanide had no effect on the SCC. In separate experiments the effect of 10(-5) M bumetanide on the O2 consumption was measured in a stirrer bath assembly. Bumetanide decreased the O2 consumption from 352 +/- 14 to 297 +/- 19 microliter/h.cm2 (significantly different from sham-treated controls). This decrease was similar to that obtained with furosemide or when Cl was removed from the bathing medium. We infer from these results that bumetanide is a selective inhibitor of active Cl transport in the bullfrog cornea.

Action of adenosine on chloride active transport of isolated frog cornea

1979 ◽  
Vol 237 (2) ◽  
pp. F121-F127
Author(s):  
B. S. Spinowitz ◽  
J. A. Zadunaisky
Keyword(s):  
Rana Catesbeiana ◽  
Chloride Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Adenyl Cyclase ◽  
Naturally Occurring ◽  
Chloride Pump ◽  
Net Flux ◽  
The One ◽  
Sustained Increase

Addition of adenosine (10–7 to 10–4 M) to the tear side of isolated corneas (Rana catesbeiana) produced a rapid, sustained increase in short-circuit current, potential difference, and radioisotopic chloride net flux. The increased net chloride flux accounted for the increased short-circuit current. Adenosine, a known activator of adenyl cyclase in other tissues, exerted its effects on chloride transport through a receptor different from the one described for epinephrine and prostaglandins in the corneal epithelium. Propranolol inhibited the epinephrine response but not the adenosine effect. Dipolyphloretin phosphate inhibited prostaglandin responses but did not affect the adenosine stimulation of chloride transport. Adenine and/or ribose, parts of the adenosine molecule, had no stimulatory effect, but 5'-AMP had a partial effect.The activation of the chloride pump with DBcAMP blocked the response to adenosine. Adenosine interacted with the effects of theophylline. Adenosine, a naturally occurring molecule, stimulated chloride transport by activation of adenyl cyclase through a separate membrane receptor in the corneal eqithelium.

Effect of acetazolamide on sodium and chloride transport by in vitro rabbit ileum

1975 ◽  
Vol 228 (6) ◽  
pp. 1808-1814 ◽  
Author(s):  
HN Nellans ◽  
RA Frizzell ◽  
SG Schultz
Keyword(s):  
Cyclic Amp ◽  
Chloride Transport ◽  
Short Circuit ◽  
Electrical Potential ◽  
Direct Interaction ◽  
Short Circuit Current ◽  
Electrical Potential Difference ◽  
Membrane Component ◽  
Rabbit Ileum

Acetazolamide (8 mM) aboishes active Cl absorption and inhibits but does not abolish active Na absorption by stripped, short-circuited rabbit ileum. These effects are not accompanied by significant changes in the transmural electrical potential difference or short-circuit current. Studies of the undirectional influxes of Na andCl indicate that acetazolamide inhibits the neutral, coupled NaCl influx process at the mucosal membranes. This action appears to explain the observed effect of acetazolamide on active, transepithelial Na and Cl transport. Acetazolamide did not significantly inhibit either spontaneous or theophylline-induced Cl secretion by this preparation, suggesting that the theophylline-induced secretion may not simply be due tothe unmasking of a preexisting efflux process when the neutral influx mechanism is inhibited by theophylline. Finally, inhibition of the neutral NaCl influx process by acetazolamide does not appear to be attributable to an inhibition of endogenous HCO3production or an elevation in intracellular cyclic-AMP levels. Instead, it appearstheat the effect of acetazolamide is due to a direct interaction with a membrane component involved in the coupled influx process.

Mechanisms and properties of sodium transport in locust rectum

1987 ◽  
Vol 65 (12) ◽  
pp. 3084-3092 ◽  
Author(s):  
K. Black ◽  
J. Meredith ◽  
B. Thomson ◽  
J. Phillips ◽  
T. Dietz
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Water Soluble ◽  
Neurosecretory System ◽  
Apical Plasma Membrane ◽  
Organic Substrates ◽  
Active Process ◽  
Soluble Factors ◽  
Flux Formula ◽  
Net Flux

Steady-state fluxes of 22Na+ were measured as a function of external Na+ concentration across locust recta mounted as flat sheets under short-circuit conditions. A net flux [Formula: see text] at all concentrations confirmed that absorption was an active process obeying Michaelis–Menten kinetics with Kt of 17 mM and Vmax of 1.5 to 2.5 μequiv. cm−2 h−1. [Formula: see text] was not affected by cAMP, was independent of external Cl− levels and stimulated short-circuit current (i.e., Cl− transport), and was partially inhibited by amiloride and ouabain but not by vanadate. Using intracellular electrodes, a small Na+ conductance that was not due to cotransport with organic substrates was identified in the apical plasma membrane. A semiquantitative model for Na+ absorption in this epithelium is proposed. We surveyed the locust neurosecretory system for water-soluble factors that might control rectal Na+ reabsorption.

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