Cyclic AMP-induced mucin exocytosis is independent of Cl− movements in human colonic epithelial cells (HT29-Cl.16E)

The human colonic epithelial goblet cell line HT29-Cl.16E was used to test whether stimulated Cl- transport is involved in the mucin exocytotic response to an increase in intracellular cyclic AMP by measuring in parallel the short-circuit current (Isc) and mucin exocytosis. Addition of 50 microM forskolin to HT29-Cl.16E cells resulted in a 2-fold stimulation of mucin release and an increase in Isc by 20 microA/cm2. To evaluate the requirement for cosecretion of Cl-, the Cl- flux was altered by three different manipulations: (1) Cl- in the medium was replaced by the poorly transported anion gluconate; (2) basolateral Cl- influx through the Na(+)-K(+)-2Cl- cotransporter was inhibited by bumetanide; and (3) an inward Cl- flux through the apical plasma membrane was generated by reversing the Cl- gradient. These manipulations did not change the forskolin-stimulated mucin release and thereby provide evidence that Cl- movements are not required for fusion of mucin granules with the plasma membrane.

Download Full-text

Metabolic Support of Chloride-dependent Short-circuit Current Across Locust Rectum

Journal of Experimental Biology ◽

10.1242/jeb.99.1.349 ◽

1982 ◽

Vol 99 (1) ◽

pp. 349-362

Author(s):

M. CHAMBERLIN ◽

J. E. PHILLIPS

Keyword(s):

Cyclic Amp ◽

Short Circuit ◽

Malpighian Tubule ◽

Short Circuit Current ◽

Metabolic Substrates ◽

Endogenous Substrates ◽

Tubule Fluid ◽

Substrate Source ◽

Stimulation Of

1. Recta of desert locusts were short-circuited and depleted of endogenous substrates by exposing them to saline containing cyclic AMP but no metabolites. Individual substrates were then added to substrate-depleted recta and the change in short-circuit current (Isc) monitored. 2. Proline or glucose (50 mM) caused by far the largest increase in Isc of all substrates tested. Stimulation of the Isc by proline was not dependent upon external sodium, but did require external chloride. 3. Physiological levels of proline also caused a large increase in Isc, while physiological levels of glucose produced a much smaller stimulation. Over 90% of the proline-dependent Isc stimulation can be produced by adding 15 mM proline solely to the lumen side of the tissue. 4. These results are discussed with regard to rectal oxidative metabolism and availability of metabolic substrates in vivo. High levels of proline in Malpighian tubule fluid are probably the major substrate source for rectal Cl−transport. Note:

Download Full-text

Acid-Base Transport and Control in Locust Hindgut: Artefacts Caused by Short-Circuit Current

Journal of Experimental Biology ◽

10.1242/jeb.155.1.455 ◽

1991 ◽

Vol 155 (1) ◽

pp. 455-467

Author(s):

R. BRENT THOMSON ◽

N. AUDSLEY ◽

JOHN E. PHILLIPS

Keyword(s):

Cyclic Amp ◽

Acid Secretion ◽

Short Circuit ◽

Short Circuit Current ◽

Salt Bridges ◽

Electrical Parameters ◽

Acid Base ◽

Before And After ◽

And Control ◽

Stimulation Of

The commonly used method of passing short-circuit current (Isc) across insect epithelia through Ag-AgCl electrodes, without the use of salt bridges, leads to significant OH− production at the cathode (lumen side) when high currents are applied. The alkalization of the lumen previously reported when cyclic AMP was added to short-circuited locust hindgut is a result of this phenomenon rather than cyclic-AMP-mediated stimulation of acid-base transport in the hindgut. When salt bridges are used to pass short-circuit current across locust hindgut, acid secretion (JH) into the lumen equals alkaline movement (JOH) to the haemocoel side, and JH is similar under both open- and short-circuit conditions. JH is similar (1.5 μequiv cm−2 h−1) in recta and ilea. Addition of cyclic AMP inhibits JH across the rectum by 42–66%, but has no effect on the ileum when salt bridges are used. Electrical parameters (Isc, Vt, Rt) reflecting hindgut Cl− transport (JCL) before and after stimulation with cyclic AMP are the same whether or not salt bridges are used. We found no evidence of any coupling between JCl and JH/JOH.

Download Full-text

Nucleotides regulate NaCl transport in mIMCD-K2 cells via P2X and P2Y purinergic receptors

AJP Renal Physiology ◽

10.1152/ajprenal.1999.277.4.f552 ◽

1999 ◽

Vol 277 (4) ◽

pp. F552-F559 ◽

Cited By ~ 37

Author(s):

David E. McCoy ◽

Amanda L. Taylor ◽

Brian A. Kudlow ◽

Katherine Karlson ◽

Margaret J. Slattery ◽

...

Keyword(s):

Plasma Membrane ◽

Apical Membrane ◽

Collecting Duct ◽

Short Circuit ◽

Short Circuit Current ◽

Extracellular Nucleotides ◽

P2x Receptor ◽

Apical Plasma Membrane ◽

Receptor Subtypes ◽

Nacl Transport

Extracellular nucleotides regulate NaCl transport in some epithelia. However, the effects of nucleotide agonists on NaCl transport in the renal inner medullary collecting duct (IMCD) are not known. The objective of this study was to determine whether ATP and related nucleotides regulate NaCl transport across mouse IMCD cell line (mIMCD-K2) epithelial monolayers and, if so, via what purinergic receptor subtypes. ATP and UTP inhibited Na+ absorption [measured via Na+ short-circuit current[Formula: see text])] and stimulated Cl− secretion [measured via Cl−short-circuit current ([Formula: see text])]. Using selective P2 agonists, we report that P2X and P2Y purinoceptors regulate [Formula: see text] and[Formula: see text]. By RT-PCR, two P2X receptor channels (P2X3, P2X4) and two P2Y G protein-coupled receptors (P2Y1, P2Y2) were identified. Functional localization of P2 purinoceptors suggest that [Formula: see text] is stimulated by apical membrane-resident P2Y purinoceptors and P2X receptor channels, whereas[Formula: see text] is inhibited by apical membrane-resident P2Y purinoceptors and P2X receptor channels. Together, we conclude that nucleotide agonists inhibit[Formula: see text] across mIMCD-K2 monolayers through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane, whereas extracellular nucleotides stimulate [Formula: see text]through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane.

Download Full-text

Studies on Locust Rectum: I. Stimulants of Electrogenic Ion Transport

Journal of Experimental Biology ◽

10.1242/jeb.86.1.211 ◽

1980 ◽

Vol 86 (1) ◽

pp. 211-223

Author(s):

J. H. SPRING ◽

J. E. PHILLIPS

Keyword(s):

Steady State ◽

Cyclic Amp ◽

Short Circuit ◽

Short Circuit Current ◽

Lag Time ◽

Camp Concentration ◽

Maximum Increase ◽

Electrogenic Ion Transport ◽

The Relationship ◽

Stimulation Of

1. Homogenates of whole corpora cardiaca (CC) cause increases in the short-circuit current (Isc) and transepithelial electropotential difference (PD) across locust recta of 3-fold and 1.7-fold respectively, in comparison with the values for unstimulated steady-state recta. Maximum stimulation restores rectal ISC and PD to levels observed immediately after removing this organ from animals. 2. Cyclic-AMP causes a similar maximum increase in ISC and PD; however, the response exhibits a much shorter lag time and a faster rate of rise than is observed for stimulation with CC. 3. The addition of CC to the haemocoel side of everted rectal sacs caused whole tissue levels of cAMP in this organ to increase 3-fold. 4. The relationship between the logarithm of CC or cAMP concentration and the increase in ISC is linear, and the decline in ΔISC with time is also dosedependent. 5. Small maximum increases in ISC are caused by homogenates of ventral ganglia, whole brain and rectal tissue, but the concentration of the stimulatory activity in these locust tissues is clearly three orders of magnitude lower than in CC. 6. Inhibitors of HCO3—/H+ and Cl− transport in vertebrate systems, acetazolamide and thiocyanate, do not inhibit the stimulation of recta by CC or cAMP.

Download Full-text

Antisecretory effects of berberine in rat ileum

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1981.241.3.g253 ◽

1981 ◽

Vol 241 (3) ◽

pp. G253-G258 ◽

Cited By ~ 1

Author(s):

Y. H. Tai ◽

J. F. Feser ◽

W. G. Marnane ◽

J. F. Desjeux

Keyword(s):

Cyclic Amp ◽

Adenylate Cyclase ◽

Short Circuit ◽

Short Circuit Current ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Rat Ileum ◽

Ion Secretion ◽

Stimulation Of

The in vitro antisecretory effects of the alkaloid berberine (1.0 mM) on intestinal ion secretion and mucosal adenylate cyclase and Na-K-ATPase activities were studied in the rat ileum. Mucosal berberine did not alter the individual basal net ion fluxes and basal adenylate cyclase activity but decreased short-circuit current (Isc) and increased the net absorption of chloride plus bicarbonate. In the cholera toxin-treated tissue, mucosal berberine stimulated absorption of Na and Cl and inhibited the increased adenylate cyclase activity but did not change the specific Na-K-ATPase activity, whereas serosal berberine stimulated Na secretion and decreased Isc. Mucosal berberine also decreased Isc, increased Cl permeability, and reversed the ion secretion induced by dibutyryl cyclic AMP, the heat-stable enterotoxin of Escherichia coli, and methylprednisolone administration. The antisecretory effects of mucosal berberine may be explained by stimulation of a Na-Cl-coupled absorptive transport process. The mechanism of action of serosal berberine remains to be elucidated. However, it is clear that mucosal berberine affects intestinal ion transport by mechanisms different from stimulation of the Na pump and probably at a step distal to the production or degradation of cyclic AMP or cyclic GMP.

Download Full-text

CYCLIC AMP STIMULATION OF ELECTROGENIC UPTAKE OF Na+ AND Cl- ACROSS THE GILL EPITHELIUM OF THE CHINESE CRAB ERIOCHEIR SINENSIS

Journal of Experimental Biology ◽

10.1242/jeb.188.1.159 ◽

1994 ◽

Vol 188 (1) ◽

pp. 159-174 ◽

Cited By ~ 2

Author(s):

S Riestenpatt ◽

W Zeiske ◽

H Onken

Keyword(s):

Cyclic Amp ◽

Electromotive Force ◽

Single Channel ◽

Short Circuit ◽

Ussing Chamber ◽

Eriocheir Sinensis ◽

Na Channel ◽

Before And After ◽

Gill Lamellae ◽

Stimulation Of

Split gill lamellae (epithelium plus cuticle) of hyperregulating Chinese crabs acclimated to fresh water were mounted in a modified Ussing chamber. Active and electrogenic absorption of sodium and chloride were measured as positive amiloride-sensitive and negative Cl--dependent short-circuit currents (INa, ICl), respectively. Both currents were characterized before and after treatment of the tissue with theophylline or dibutyryl cyclic AMP. Both drugs increased INa and ICl. A simple circuit analysis showed that INa stimulation reflected a marked increase in the transcellular Na+ conductance, whereas the respective electromotive force was unchanged. The Michaelis constant (KNa) for Na+ current saturation was decreased after INa stimulation, indicating an increased affinity of the transport mechanism for its substrate. Consequently, the affinity for the Na+ channel blocker amiloride decreased as expected for a competitive interaction between substrate and inhibitor. Analysis of the amiloride-induced current-noise revealed a marked increase in the number of apical Na+ channels after INa stimulation with theophylline, whereas there was little change in the single-channel current. Stimulation of Cl- absorption was accompanied by a substantial increase in both transcellular conductance and electromotive force, indicating an activation of the apical H+ pump that provides the driving force for active Cl- uptake via apical Cl-/HCO3- exchange and basolateral Cl- channels.

Download Full-text

Active Transport by the Cecropia Midgut

Journal of Experimental Biology ◽

10.1242/jeb.48.1.1 ◽

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.

Download Full-text

Steroid hormone stimulation of Na+ transport in A6 cells is mediated via glucocorticoid receptors

AJP Cell Physiology ◽

10.1152/ajpcell.1993.264.4.c875 ◽

1993 ◽

Vol 264 (4) ◽

pp. C875-C884 ◽

Cited By ~ 46

Author(s):

T. J. Schmidt ◽

R. F. Husted ◽

J. B. Stokes

Keyword(s):

Cell Line ◽

Glucocorticoid Receptors ◽

Short Circuit ◽

Short Circuit Current ◽

Mineralocorticoid Receptors ◽

Na Transport ◽

Receptor Complexes ◽

Binding Forms ◽

Mineralocorticoid Antagonists

The A6 cell line derived from the toad kidney forms polarized, highly differentiated epithelial monolayers in culture and has been utilized as an experimental model for studying regulation of transepithelial Na+ transport by aldosterone. In the present study we evaluated the specific role(s) of glucocorticoid and mineralocorticoid receptors in mediating this enhanced electrogenic Na+ transport, which was measured experimentally as an increase in short-circuit current (Isc). Our data demonstrate that specific glucocorticoid agonists (100 nM), including RU 28362 and RU 26988, elicit “mineralocorticoid-like” increases in Isc that are blocked by the glucocorticoid antagonist RU 38486 but are unaffected by mineralocorticoid antagonists including RU 28318 and RU 26752. The stimulatory effects of aldosterone (100 nM) were also blocked by RU 38486 and not by mineralocorticoid antagonists. These data extend earlier studies suggesting that in this cell line aldosterone mediates its physiological effects via binding with relatively low affinity (dissociation constant Kd congruent to 25-50 nM) to glucocorticoid receptors, despite the presence of apparently normal mineralocorticoid receptors. Our in vitro biochemical studies also demonstrate that A6 glucocorticoid receptor complexes can be thermally activated or transformed to DNA binding forms which exhibitaltered elution profiles from anion-exchange resins. Thus, based on several criteria, these amphibian glucocorticoid receptors appear very similar to classical mammalian receptors and are capable of mediating all of the stimulatory effects of aldosterone on net Na+ transport.

Download Full-text

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

AJP Cell Physiology ◽

10.1152/ajpcell.1990.259.2.c215 ◽

1990 ◽

Vol 259 (2) ◽

pp. C215-C223 ◽

Cited By ~ 6

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)

Download Full-text

Immunological regulation of colonic ion transport

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1989.256.2.g396 ◽

1989 ◽

Vol 256 (2) ◽

pp. G396-G403 ◽

Cited By ~ 4

Author(s):

D. A. Russell ◽

G. A. Castro

Keyword(s):

Guinea Pigs ◽

Trichinella Spiralis ◽

Short Circuit ◽

Short Circuit Current ◽

Antigenic Stimulation ◽

Synthesis Inhibitor ◽

Combined Use ◽

Maximum Elevation ◽

Prostaglandin Synthesis Inhibitor ◽

Stimulation Of

Challenge of distal colonic epithelium from Trichinella spiralis-infected guinea pigs with parasite-derived antigen elevated short-circuit current (Isc) for approximately 60 min. The maximum elevation (delta Isc) was approximately 250 microA/cm2 at 5 min after the addition of trichinella antigen. The antigen-induced alterations in Isc were of greater magnitude and duration than those evoked in jejunum. Colonic electrical resistance was transiently reduced after exposure to antigen. There was no significant effect of antigen on electrical parameters of colon from nonimmunized (uninfected) guinea pigs. The antihistamine pyrilamine (10(-5) M) and the prostaglandin synthesis inhibitor indomethacin (10(-6) M) reduced the colonic Isc response to antigen by 40% when used in combination but had insignificant effects when used singly. In contrast, the jejunal Isc response to antigen was totally eliminated by the combined use of those inhibitors. Antigenic stimulation of sensitized colon released histamine and prostaglandin E2 (PGE2). However, the histamine released was only about one-tenth that stimulated by antigen in the jejunum, and PGE2 released was only one-tenth of that stimulated by bradykinin in the colon. PGE2 was not released after antigenic stimulation of jejunum. The antigen-induced colonic delta Isc was reduced approximately 50% by either furosemide or tetrodotoxin. Although histamine- and indomethacin-sensitive factors contribute greatly to the mediation of the antigen-induced delta Isc in jejunum, these autacoids contribute to a lesser extent to the antigen-induced delta Isc in guinea pig colon.

Download Full-text