Recruitment of purinergically stimulated Cl- channels from granule membrane to plasma membrane

1996 ◽  
Vol 271 (2) ◽  
pp. C612-C619 ◽  
Author(s):  
D. Merlin ◽  
X. Guo ◽  
K. Martin ◽  
C. Laboisse ◽  
D. Landis ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Lines ◽  
Inhibitory Effects ◽  
Fungal Metabolite ◽  
Cl Secretion ◽  
Fusion Inhibitors ◽  
Granule Membrane ◽  
Cl Channels ◽  
Ht 29 ◽  
Cl Conductance

HT29-Cl.16E and HT29-Cl.19A are two different subcloned cell lines derived from the human adenocarcinoma cell line HT-29. They are similar in their ability to grow and differentiate to polarized epithelial cells but differ in that HT29-Cl.16E is goblet cell-like with many mucin granules, whereas HT29-Cl.19A lacks mucin granules. Extracellular ATP stimulates Cl- secretion in both cell lines through luminal purinergic P20 receptors and, in HT29-Cl.16E, also mucin secretion release. To evaluate whether fusion of mucin granules is associated with an increase in Cl- conductance of the plasma membrane, the effects of two fusion inhibitors on luminal Cl- conductance were measured. Blockage of actin depolymerization with phalloidin (1 microM) inhibited purinergically stimulated but not adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated luminal Cl- efflux by 50% in HT29-Cl.16E. The same treatment was without effect in HT29-Cl.19A. The fungal metabolite wortmannin, which is an inhibitor of regulated exocytosis in leukocytes, at 100 nM inhibited Cl- secretion by 70% in HT29-Cl.16E. This inhibition was not a direct effect on purinergically stimulated Cl- channels because wortmannin concentrations of up to 1 microM did not affect the secretory response in HT29-Cl.19A. The wortmannin inhibition of Cl- secretion is associated with an inhibition of granule fusion as judged by electron microscopy. The differential inhibition of Cl- secretion in the related HT-29 clones that differ with respect to the presence of mucin granules indicates that 1) the granule fusion inhibitors, phalloidin and wortmannin, have no direct inhibitory effects on purinergically and cAMP-activated Cl- channels, 2) a major portion of purinergically but not cAMP-activated Cl- channels is associated with granule fusion in HT29-Cl.16E, and 3) the signaling pathways for Cl- secretion and granule fusion are not completely identical.

Psoralens: novel modulators of Cl- secretion

1997 ◽  
Vol 272 (3) ◽  
pp. C976-C988 ◽  
Author(s):  
D. C. Devor ◽  
A. K. Singh ◽  
R. J. Bridges ◽  
R. A. Frizzell
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Receptor Activation ◽  
Sustained Response ◽  
Rat Colon ◽  
Cl Secretion ◽  
Cl Channel ◽  
Cl Channels ◽  
Cl Conductance

We evaluated effects of psoralens on Cl- secretion (short-circuit current, I(sc)) across T84 monolayers. Methoxsalen failed to increase I(sc). Several observations suggest that psoralens open cystic fibrosis transmembrane conductance regulator Cl- channels. 1) After activation of the Ca2+-dependent basolateral membrane K+ channel (K(Ca)) by 1-ethyl-2-benzimidazolinone or thapsigargin, methoxsalen (10 microM) further increased I(sc). 2) When added before carbachol (CCh), methoxsalen potentiated the I(sc) response to CCh, as predicted, if it increased apical Cl- conductance. 3) After establishment of a mucosal-to-serosal Cl- gradient and permeabilization of basolateral membrane with nystatin, psoralens increased Cl- current, which was inhibited by glibenclamide. In contrast, neither TS-TM calix[4]arene nor Cd2+, inhibitors of outwardly rectifying Cl- channels and the ClC-2 Cl-channel, respectively, inhibited psoralen-induced Cl- current. In contrast to their effects on Cl- conductance, psoralens failed to significantly affect basolateral membrane K+ conductance; subsequent addition of 1-ethyl-2-benzimidazolinone induced a large increase in K+ conductance. Also, in excised patches, methoxsalen failed to activate K(Ca). In addition to potentiating the peak response to CCh, psoralens induced a secondary, sustained response. Indeed, when added up to 60 min after return of CCh-induced I(sc) to baseline, psoralens induced a sustained I(sc). This sustained response was inhibited by atropine, demonstrating the requirement for continuous muscarinic receptor activation by CCh. This sustained response was inhibited also by verapamil, removal of bath Ca2+, and charybdotoxin. These results suggest that return of I(sc) to baseline after CCh stimulation is not due to downregulation of Ca2+ influx or K(Ca). Finally, we obtained similar results with psoralens in rat colon and primary cultures of murine tracheal epithelium. On the basis of these observations, we conclude that psoralens represent a novel class of Cl- channel openers that can be used to probe mechanisms underlying Ca2+-mediated Cl- secretion.

Inhibition of cAMP- and Ca-dependent Cl- secretion by phorbol esters: inhibition of basolateral K+ channels

1993 ◽  
Vol 264 (1) ◽  
pp. C161-C168 ◽  
Author(s):  
W. W. Reenstra
Keyword(s):  
Phorbol Esters ◽  
Short Circuit ◽  
Short Circuit Current ◽  
T84 Cells ◽  
K Channels ◽  
Cl Secretion ◽  
Cyclic Monophosphate ◽  
Regulator Protein ◽  
Cl Channels ◽  
Cl Conductance

Pretreating confluent T84 cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA) inhibits adenosine 3',5'-cyclic monophosphate (cAMP)- and carbachol-induced Cl secretion. Both a sustained short-circuit current (Isc), seen after the addition of 50 microM 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) and 100 microM 3-isobutyl-1-methylxanthine (IBMX), and a transient current, seen after the subsequent addition of 100 microM carbachol, are inhibited by 80% following pretreatment with 100 nM PMA for 2 h. Pretreatment with PMA has no effect on the level of cystic fibrosis transmembrane conductance regulator protein or apical cAMP-dependent Cl conductance. Carbachol does not induce an increase in apical Cl conductance. Basolateral K conductance was measured in monolayers treated with apical nystatin and exposed to a K gradient. Agonist-independent K conductance is 10-fold greater in Cl media than in gluconate media. Pretreatment with PMA inhibits agonist-independent K conductance by 57% in Cl media but stimulates K conductance by 1.9-fold in gluconate media. The addition of carbachol induces a transient increase in basolateral K conductance, and pretreatment with PMA inhibits the agonist-dependent K conductance by 66% in Cl media and by 92% in gluconate media. In Cl media, serosal barium, at 3 mM, inhibits agonist-independent K conductance but has no significant effect on the carbachol-induced conductance. In nonpermeabilized monolayers, serosal barium inhibits the cAMP-dependent Isc by 56% but has no effect on the carbachol-induced Isc. These results demonstrate that the primary effect of PMA on Cl secretion is not inhibition of apical Cl channels but inhibition of basolateral K channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Polarization-dependent apical membrane CFTR targeting underlies cAMP-stimulated Cl- secretion in epithelial cells

1994 ◽  
Vol 266 (1) ◽  
pp. C254-C268 ◽  
Author(s):  
A. P. Morris ◽  
S. A. Cunningham ◽  
A. Tousson ◽  
D. J. Benos ◽  
R. A. Frizzell
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Brefeldin A ◽  
Microtubule Organizing Center ◽  
Cl Secretion ◽  
Organizing Center ◽  
Reversible Time ◽  
Golgi Network ◽  
Ht 29 ◽  
Cl Conductance

The relationship between adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion and the cellular location of the cystic fibrosis transmembrane conductance regulator (CFTR) was determined in both polarized (Cl.19A) and unpolarized (parental) HT-29 colonocytes expressing similar levels of CFTR mRNA and protein. CFTR immunolocalized to the apical membrane domain of polarized colonocytes exhibiting cAMP-responsive Cl- secretion. In contrast, CFTR staining was perinuclear in unpolarized colonocytes, which gave little or no cAMP-stimulated Cl- conductance responses. Thus cAMP-stimulated Cl- secretion coincided with an apical localization of CFTR. Brefeldin A (BFA) was used to perturb glycoprotein targeting in these cells. In polarized colonocytes, BFA caused a reversible, time-dependent decrease in the Cl-conductance response to cAMP but not Ca2+. Apical CFTR redistributed into large coalesced intracellular vesicles, located within the same plane as the microtubule organizing center, a marker for the trans-Golgi network (TGN). In preconfluent monolayers or unpolarized HT-29 cells, BFA had no effect on CFTR staining, which remained perinuclear. Mature, Golgi-processed CFTR protein was isolated from both polarized and unpolarized colonocytes. Thus the mechanism for polarization-dependent apical membrane CFTR targeting and the acquisition of cAMP-dependent Cl- secretion lies at or beyond the late Golgi-TGN in epithelial cells.

Long-term cAMP activation of Na(+)-K(+)-2Cl- cotransporter activity in HT-29 human adenocarcinoma cells

1993 ◽  
Vol 264 (4) ◽  
pp. C857-C865 ◽  
Author(s):  
I. N. Slotki ◽  
W. V. Breuer ◽  
R. Greger ◽  
Z. I. Cabantchik
Keyword(s):  
Cystic Fibrosis ◽  
Cell Lines ◽  
De Novo ◽  
Transport Systems ◽  
Short Term ◽  
Cl Channel ◽  
Epithelial Cell Lines ◽  
Cl Channels ◽  
Ht 29

Cl- channel and Na(+)-K(+)-2Cl- cotransport activities were studied in various cystic fibrosis transmembrane conductance regulator (CFTR)-expressing cells with the aim of assessing integrative patterns of regulation of Cl- secretion. Human colonic HT-29 cells express relatively high levels of CFTR and cotransporter but relatively low Cl- channel activity. These cells showed commensurate activations of both transport systems evoked by short-term (minutes) or long-term (hours) exposures to adenosine 3',5'-cyclic monophosphate (cAMP). However, unlike in the case of CFTR and Cl- channels, long-term induction of cotransporter did not depend on de novo protein synthesis or changes in number of transporters. The patterns of activation of both transporters were also examined in CFTR-deficient cell lines (CFPAC and the viral-transfected CFPAC-PLJ) and in the viral CFTR-transfected derivative (CFPAC-4.7). All these cells displayed relatively low basal cotransport activity and a correspondingly low number of transporters, whereas only CFPAC-4.7 cells showed short-term (but not long-term) activatable Cl- channels. However, irrespective of the presence or absence of CFTR in CFPAC cells, neither short- nor long-term cAMP exposures induced significant cotransporter activation. Our studies with the various epithelial cell lines indicate that expression of CFTR activity per se is not sufficient for stimulation of cotransporter activity. Moreover, despite apparent correction of CFTR levels in CFPAC cells by gene transfer, the apparent Cl- secretory capacity might be limited by the low cotransport activity, such as that found in CFPAC cells, with obvious implications for proposed gene therapy of cystic fibrosis.

Selection for MDR1/P-glycoprotein enhances swelling-activated K+ and Cl- currents in NIH/3T3 cells

1994 ◽  
Vol 267 (2) ◽  
pp. C650-C658 ◽  
Author(s):  
D. B. Luckie ◽  
M. E. Krouse ◽  
K. L. Harper ◽  
T. C. Law ◽  
J. J. Wine
Keyword(s):  
Cell Lines ◽  
Response Curve ◽  
Drug Transport ◽  
Regulatory Volume Decrease ◽  
Absolute Difference ◽  
P Glycoprotein ◽  
Cl Channels ◽  
Selection For ◽  
Nih 3T3 ◽  
Cl Conductance

The relationship between multidrug resistance (MDR) P-glycoprotein expression and swelling-activated Cl- and K+ conductance was investigated in mouse NIH/3T3 fibroblasts and their colchicine-selected counterparts (COL1000, high P-glycoprotein). Whole cell patch-clamp and isotopic flux experiments confirmed that swelling-activated Cl- currents were induced by 20-30% bath dilution only in the MDR-expressing cell line. However, at bath dilutions > 30%, both cell lines developed Cl- currents that reached similar large magnitudes at higher dilution levels. Thus the apparent absolute difference in cell lines at lower dilutions is due to a shift in the response curve relating hypotonicity to Cl- conductance. At all dilutions and in both cell lines, the swelling-activated Cl- currents were outwardly rectifying, active at negative cell voltages, and inactivated at positive voltages. Verapamil (100 microM) and 1,9-dideoxyforskolin (100 microM), which inhibit P-glycoprotein drug transport, did not significantly inhibit the swelling-activated Cl- conductance efflux in the COL1000 cells also showed a leftward shift in the response curve to hypotonicity. These results indicate that response curve to hypotonicity. These results indicate that colchicine-selection for increased P-glycoprotein expression did not lead to the expression of swelling-activated Cl- channels, but instead enhanced a step in the pathway from bath dilution to regulatory volume decrease that is common to both K+ and Cl- channels.

scholarly journals Simultaneous analysis of cell Ca2+ and Ca2(+)-stimulated chloride conductance in colonic epithelial cells (HT-29).

1990 ◽  
Vol 1 (12) ◽  
pp. 951-963 ◽  
Author(s):  
A P Morris ◽  
K L Kirk ◽  
R A Frizzell
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Inhibitory Pathway ◽  
Colonic Epithelial Cells ◽  
Chloride Currents ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging ◽  
Cell Current ◽  
Ht 29 ◽  
Cl Conductance

We used perforated patch, whole-cell current recordings and video-based fluorescence ratio imaging to monitor the relation of plasma membrane ionic conductances to intracellular free Ca2+ within individual colonic epithelial cells (HT-29). The Ca2(+)-mediated agonist, neurotensin, activated K+ and Cl- conductances that showed different sensitivities to [Ca2+]i. The Cl- conductance was sensitive to increases or decreases in [Ca2+]i around the resting value of 76 +/- 32 (mean +/- SD) nM (n = 46), whereas activation of the K+ conductance required at least a 10-fold rise in [Ca2+]i. Neurotensin increased [Ca2+]i by stimulating a transient intracellular Ca2+ release, which was followed by a sustained rise in [Ca2+]i due to Ca2+ influx from the bath. The onset of the initial [Ca2+]i transient, monitored at a measurement window over the cell interior, lagged behind the rise in Cl- current during agonist stimulation. This lag was not present when the [Ca2+]i rise was due to Ca2+ entry from the bath, induced either by the agonist or by the Ca2+ ionophore ionomycin. The temporal differences in [Ca2+]i and Cl- current during the agonist-induced [Ca2+]i transient can be explained by a localized Ca2+ release from intracellular stores in the vicinity of the plasma membrane Cl- channel. Chloride currents recover toward basal values more rapidly than [Ca2+]i after the agonist-induced [Ca2+]i transient, and, during a sustained neurotensin-induced [Ca2+]i rise, Cl- currents inactivate. These findings suggest that an inhibitory pathway limits the increase in Cl- conductance that can be evoked by agonist. Because this Cl- current inhibition is not observed during a sustained [Ca2+]i rise induced by ionomycin, the inhibitory pathway may be mediated by another agonist-induced messenger, such as diacylglycerol.

Cytotoxic Prenylated Xanthones from the Leaves of Garcinia bracteata

Planta Medica ◽  
2019 ◽  
Vol 85 (06) ◽  
pp. 444-452 ◽  
Author(s):  
Bao-Jun Zhang ◽  
Wen-Wei Fu ◽  
Rong Wu ◽  
Jin-Ling Yang ◽  
Cai-Yun Yao ◽  
...  
Keyword(s):  
Data Analysis ◽  
Cell Growth ◽  
Tumor Cell ◽  
Cell Lines ◽  
Spectroscopic Data ◽  
Tumor Cell Growth ◽  
Inhibitory Effects ◽  
Inhibitory Activities ◽  
Ht 29

Six new prenylated xanthones (1–6) and seventeen known xanthones were isolated from extracts of Garcinia bracteata leaves. Their structures were determined by extensive NMR and MS spectroscopic data analysis. The inhibitory activities of the isolates were assayed on HeLa, A549, PC-3, HT-29, and WPMY-1 cell lines. Compounds 1 and 15–17 showed moderate inhibitory effects on tumor cell growth, with IC50s ranging from 3.7 to 14.7 µM.

scholarly journals Actaticas A−G, Cycloartane Triterpenes From Actaea asiatica With Their Antiproliferative Activity

2021 ◽  
Vol 9 ◽  
Author(s):  
Meigeng Hu ◽  
Dan Zhao ◽  
Xudong Xu ◽  
Guoxu Ma ◽  
Haifeng Wu ◽  
...  
Keyword(s):  
Chemical Analysis ◽  
Cell Lines ◽  
Antiproliferative Activity ◽  
Inhibitory Effects ◽  
Phytochemical Studies ◽  
Ht 29 ◽  
Mcf 7

Phytochemical studies on the rhizomes of Actaea asiatica led to the isolation of seven new cycloartane triterpenes, actaticas A−G (1−7). Their structures were determined by NMR, HRESIMS, and chemical analysis. All the isolates were evaluated for their antiproliferative activity against HT-29 and McF-7 cell lines. The results showed that all the compounds displayed cytotoxicity. All compounds showed significant inhibitory effects with IC50 values of 9.2–26.4 μM.

Stimulation of Cl- secretion by extracellular ATP does not depend on increased cytosolic Ca2+ in HT-29.cl16E

1995 ◽  
Vol 269 (6) ◽  
pp. C1457-C1463 ◽  
Author(s):  
X. Guo ◽  
D. Merlin ◽  
R. D. Harvey ◽  
C. Laboisse ◽  
U. Hopfer
Keyword(s):  
Protein Kinase ◽  
Purinergic Receptors ◽  
Extracellular Atp ◽  
Equilibrium Potential ◽  
Tetraacetic Acid ◽  
Whole Cell ◽  
Cl Secretion ◽  
Ht 29 ◽  
Cl Conductance ◽  
Stimulation Of

Extracellular ATP and elevated cytosolic Ca2+ concentration ([Ca2+]i) are major secretagogues for Cl- in the goblet cell-like clone cl.16E derived from colonic HT-29 cells. The involvement of [Ca2+]i as a messenger for the purinergically stimulated Cl- secretion was investigated using whole cell patch-clamp and Ussing chamber techniques, as well as [Ca2+]i measurements using fura 2-loaded cells. Under voltage-clamp conditions, the whole cell current at +50 mV was 3 +/- 1 pA/pF under unstimulated conditions. Stimulation of purinergic receptors with 200 microM extracellular ATP increased the current at +50 mV to 41 +/- 10 pA/pF, with a half-maximal effective dose (ED50) of approximately 3 microM. The current was transient, usually lasting 1-2 min, and the current-voltage relationship was approximately linear between -70 and +50 mV. Evidence that the ATP-stimulated current was carried by Cl- included 1) the reversal potential of the current closely followed the Cl- equilibrium potential, and 2) the stimulated current was absent when Cl- was removed from both bath and pipette solutions. Exposure to ATP also increased [Ca2+]i, with an ED50 of approximately 1 microM and maximal changes (at 200 microM) from baseline (71 +/- 3 nM) to 459 +/- 50 nM. The ATP-dependent Cl- conductance increase was not diminished when [Ca2+]i was clamped at 100 nM using a Ca(2+)-1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or Ca(2+)-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffering system. However, the ATP effect did require some basal level of Ca2+ because clamping [Ca2+]i at < 10 nM abolished activation of the Cl- conductance. The presence of the protein kinase A inhibitor H-89 or the protein kinase C inhibitor staurosprine did not change the ATP-activated Cl-conductance. These data demonstrate that the ATP-stimulated increase in Cl- current does not require an increase in [Ca2+]i, suggesting the involvement of either another signaling pathway or direct activation of Cl- channels by purinergic receptors.

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