Biomedical subjects

H R de Jonge

Publications and source records attributed to H R de Jonge.

At least 37 records · Page 2Linked to original sources

PubMed1996

Serum-induced membrane depolarization in quiescent fibroblasts: activation of a chloride conductance through the G protein-coupled LPA receptor.

Serum stimulation of quiescent fibroblasts leads to a dramatic depolarization of the plasma membrane; however, the identity of the active serum factor(s) and the underlying mechanism are unknown. We find that this serum activity is attributable to albumin-bound lysophosphatidic acid (LPA) acting on its own G protein-coupled receptor, and that membrane depolarization is due to activation of an anion conductance mediating Cl- efflux. This depolarizing Cl- current can also be activated by thrombin and neuropeptide receptors; it is distinct from volume-regulated Cl- currents. Activation of the Cl- current consistently follows stimulation of phospholipase C and coincides with remodelling of the actin cytoskeleton, which is regulated by the Ras-related GTPase Rho. However, the response is not due to Ca2+/protein kinase C signalling and requires neither Rho nor Ras activation. The results indicate that in quiescent fibroblasts, LPA and other G protein-coupled receptor agonists evoke membrane depolarization by activating a new type of Cl- channel through a signalling pathway that is closely associated with phosphoinositide hydrolysis, yet independent of known second messengers.

Animals↗

PubMed1996

Activation of the osmo-sensitive chloride conductance involves P21rho and is accompanied by a transient reorganization of the F-actin cytoskeleton.

Hypo-osmotic stimulation of human Intestine 407 cells rapidly activated compensatory CL- and K+ conductances that limited excessive cell swelling and, finally, restored the original cell volume. Osmotic cell swelling was accompanied by a rapid and transient reorganization of the F-actin cytoskeleton, affecting both stress fibers as well as apical ruffles. In addition, an increase in total cellular F-actin was observed. Pretreatment of the cells with recombinant Clostridium botulinum C3 exoenzyme, but not with mutant enzyme (C3-E173Q) devoid of ADP-ribosyltransferase activity, greatly reduced the activation of the osmo-sensitive anion efflux, suggesting a role for the ras-related GTPase p21rho. In contrast, introducing dominant negative N17-p21rac into the cells did not affect the volume-sensitive efflux. Cell swelling-induced reorganization of F-actin coincided with a transient, C3 exoenzyme-sensitive tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) as well as with an increase in phosphatidylinositol-3-kinase (PtdIns-3-kinase) activity. Pretreatment of the cells with wortmannin, a specific inhibitor of PtdIns-3-kinase, largely inhibited the volume-sensitive ion efflux. Taken together, our results indicate the involvement of a p21rho signaling cascade and actin filaments in the activation of volume-sensitive chloride channels.

ADP Ribose Transferases↗

PubMed1995

Isotype-specific activation of cystic fibrosis transmembrane conductance regulator-chloride channels by cGMP-dependent protein kinase II.

Type II cGMP-dependent protein kinase (cGKII) isolated from pig intestinal brush borders and type I alpha cGK (cGKI) purified from bovine lung were compared for their ability to activate the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl- channel in excised, inside-out membrane patches from NIH-3T3 fibroblasts and from a rat intestinal cell line (IEC-CF7) stably expressing recombinant CFTR. In both cell models, in the presence of cGMP and ATP, cGKII was found to mimic the effect of the catalytic subunit of cAMP-dependent protein kinase (cAK) on opening CFTR-Cl-channels, albeit with different kinetics (2-3-min lag time, reduced rate of activation). By contrast, cGKI or a monomeric cGKI catalytic fragment was incapable of opening CFTR-Cl- channels and also failed to potentiate cGKII activation of the channels. The cAK activation but not the cGKII activation was blocked by a cAK inhibitor peptide. The slow activation by cGKII could not be ascribed to counteracting protein phosphatases, since neither calyculin A, a potent inhibitor of phosphatase 1 and 2A, nor ATP gamma S (adenosine 5'-O-(thiotriphosphate)), producing stable thiophosphorylation, was able to enhance the activation kinetics. Channels preactivated by cGKII closed instantaneously upon removal of ATP and kinase but reopened in the presence of ATP alone. Paradoxically, immunoprecipitated CFTR or CF-2, a cloned R domain fragment of CFTR (amino acids 645-835) could be phosphorylated to a similar extent with only minor kinetic differences by both isotypes of cGK. Phosphopeptide maps of CF-2 and CFTR, however, revealed very subtle differences in site-specificity between the cGK isoforms. These results indicate that cGKII, in contrast to cGKI alpha, is a potential activator of chloride transport in CFTR-expressing cell types.

Adenosine Triphosphate↗

PubMed1995

Expression of cGMP-dependent protein kinase I and phosphorylation of its substrate, vasodilator-stimulated phosphoprotein, in human endothelial cells of different origin.

Previous studies demonstrated that the thrombin-induced permeability of endothelial cell monolayers is reduced by the elevation of cGMP. In the present study, the presence of cGMP-dependent protein kinase (cGMP-PK) immunoreactivity and activity in various types of human endothelial cells (ECs) and the role of cGMP-PK in the reduction of thrombin-induced endothelial permeability was investigated. cGMP-PK type I was demonstrated in freshly isolated ECs from human aorta and iliac artery as well as in cultured ECs from human aorta, iliac vein, and foreskin microvessels. Addition of the selective cGMP-PK activator 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) to these ECs caused phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), an established cGMP-PK substrate, which is localized at cell-cell contact sites of confluent ECs. cGMP-PK type I expression decreased during serial passage of ECs, which correlated with a diminished ability of 8-pCPT-cGMP to induce VASP phosphorylation. Preincubation of aorta and microvascular EC monolayers with 8-pCPT-cGMP caused a 50% reduction of the thrombin-stimulated permeability, as determined by measuring the peroxidase passage through EC monolayers on porous filters. Furthermore, the thrombin-induced rise in cytoplasmic [Ca2+]i was strongly attenuated by the cGMP-PK activator in fura 2-loaded aorta ECs. In contrast, cGMP-PK could not be demonstrated in freshly isolated and cultured human umbilical vein ECs. Incubation of umbilical vein ECs with 8-pCPT-cGMP did not cause VASP phosphorylation and had no effect on the thrombin-induced increases in cytoplasmic Ca2+ and endothelial permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Aorta↗

PubMed1994

Heat-stable enterotoxin receptor/guanylyl cyclase C is an oligomer consisting of functionally distinct subunits, which are non-covalently linked in the intestine.

Guanylyl cyclase (GC) C is a heat-stable enterotoxin (STa) receptor with a monomeric M(r) of approximately 140,000. We calculated from its hydrodynamic parameters that an active GC-C complex has a M(r) of 393,000, suggesting that GC-C is a trimer under native conditions. Both trimeric and dimeric GC-C complexes were detected by 125I-STa binding and SDS-polyacrylamide gel electrophoresis under non-reducing conditions. The GC activity and STa binding from intestinal brush border membranes comigrated in gel filtration and velocity sedimentation with recombinant GC-C. However, 125I-STa cross-linking demonstrated that STa receptors with molecular masses of 52 and 74 kDa are non-covalently attached to GC in the intestine. Radiation inactivation revealed different functional sizes for basal GC activity, STa-stimulated GC activity, and STa binding (59, 210-240, and 32-52 kDa, respectively). At low radiation doses, basal GC activity was stimulated, suggesting that GC-C is inhibited by a relatively large, probably internal structure. These results suggest that STa may activate GC-C by promoting monomer-monomer interaction (internal "dimerization") within a homotrimeric GC-C complex, and that GC-C is proteolytically modified in the brush border membrane but retains its function.

Animals↗

PubMed1994

Ca(2+)-mobilizing hormones potentiate hypotonicity-induced activation of ionic conductances in Intestine 407 cells.

Human Intestine 407 cells respond to hyposmotic stimulation by activating the conductive efflux of both Cl- and K+ (regulatory volume decrease) through pathways involving protein tyrosine phosphorylation (Tilly, B. C., N. van den Berghe, L. G. J. Tertoolen, M. J. Edixhoven, and H. R. de Jonge. J. Biol. Chem. 268: 19919-19922, 1993). Stimulation of the cells with hormones linked to the phospholipase C signaling cascade (e.g., bradykinin, histamine, or thrombin) or with the phosphotyrosine phosphatase inhibitor vanadate, potentiated the osmosensitive anion efflux by two- to threefold but did not affect anion efflux under isotonic conditions. No substantial increase in intracellular Ca2+ concentration ([Ca2+]i) was observed on mild hypotonicity-induced cell swelling. In addition, loading the cells with the intracellular Ca2+ chelator 1,2-bis(2-amino-phenoxy)ethane- N,N,N',N',-tetraacetic acid acetoxymethyl ester (BAPTA-AM) caused a partial reduction of the osmoshock-induced 125I- efflux but did not affect its potentiation by vanadate. In contrast, bradykinin transiently elevated [Ca2+]i, and its potentiation of the osmosensitive anion efflux was completely inhibited after BAPTA-AM loading. Both the Ca(2+)-mobilizing hormones as well as osmotic cell swelling rapidly triggered the phosphorylation of several proteins on tyrosine residues. However, the effects of the hormones, but not the effect of hypotonicity, on protein tyrosine phosphorylation was largely abolished in BAPTA-loaded cells. Taken together the results indicate a novel role for Ca(2+)-mobilizing hormones, although elevation of [Ca2+]i, in potentiating volume-sensitive ionic efflux even in cell types lacking the expression of Ca(2+)-activated Cl- channels in their plasma membrane.

Anions↗

PubMed1994

Determinants of mild clinical symptoms in cystic fibrosis patients. Residual chloride secretion measured in rectal biopsies in relation to the genotype.

Previous Ussing chamber measurements of secretagogue-provoked changes in short circuit current in rectal suction biopsies of cystic fibrosis (CF) patients showed that in a minority of patients chloride secretion in response to cholinergic agonists is reduced but not completely absent. To assess a possible relationship between this phenomenon and both the genotype and the phenotype, we performed Ussing chamber experiments on rectal suction biopsies of 51 CF patients. The CF mutation was identified in 89 out of 102 CF alleles. No apparent chloride secretion was found in 30 CF patients (group I). Low residual chloride secretion was found in 11 CF patients (group II), while a relatively high residual secretion appeared in 10 CF patients (group III). Pancreatic function was preserved more frequently in CF patients displaying residual secretion: 0% in group I, 27% in group II, and 60% in group III (P < 0.001). The age at diagnosis (mean +/- SEM) in group III (18.4 +/- 6.6) was significantly different from group I (1.2 +/- 0.4, P < 0.01) and group II (3.5 +/- 1.4, P = 0.05). Residual chloride secretion was found in some of the 28 dF508 homozygous patients (three in group II, and one in group III), disclosing that other factors than the CF gene defect itself affect the transepithelial chloride transport. The age at diagnosis correlates significantly with the magnitude of the secretory response, even within the dF508 homozygous patients (r = 0.4, P < 0.05). We conclude that residual chloride secretion in CF is the pathophysiological basis of preserved pancreatic function and delayed presentation of the disease, which is not exclusively determined by the CF genotype.

Adolescent↗

PubMed1994

Effect of anisotonic conditions on the transport of hydrophilic model compounds across monolayers of human colonic cell lines.

The effect of anisotonic solutions on the enhancement of the transport of hydrophilic model compounds across monolayers of Caco-2 and HT-29.cl19A intestinal epithelial cells was studied. In filter-grown monolayers of the highly differentiated villus-like Caco-2 cell line, a profound and dose-dependent drop in the transepithelial electrical resistance was found after apical treatment with a 30 or a 50% hypotonic solution (200 and 150 mOsmol, respectively). This drop was not observed after basolateral and two-sided application of a 50% hypotonic solution. During apical hypotonic treatment a 12- and 8-fold increase also was observed in transepithelial transport of two hydrophilic model compounds, i.e., fluorescein-Na and fluorescein-isothiocyanate-labeled dextran, MW 4000, respectively. Through confocal laser scanning microscopy, it was revealed that this enhanced transport was predominantly via the paracellular route. Moreover, morphological changes in the cell layers indicating cell swelling were observed after apical hypotonic, but not after basolateral or bilateral treatment, probably resulting from an incomplete regulatory volume decrease response. This swelling, and slight lateral retraction of the cells, allowed the hydrophilic compounds to pass between the cells. The effects of hypotonic challenge also were studied in monolayers of the more crypt cell-like HT-29.cl19A cell line. After apical hypotonic shock, these cells showed no effect on transepithelial electrical resistance, whereas an increase was observed after basolateral and bilateral treatment. Hypotonic shock failed to increase the transport of the hydrophilic model compounds in this cell line.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1993

Protein tyrosine phosphorylation is involved in osmoregulation of ionic conductances.

Using the human Intestine 407 cell line as a model, we investigated a possible role for tyrosine kinase(s) in regulating the ion efflux pathways induced by hyposmotic stimulation (regulatory volume decrease, RVD). Pretreatment of 125I(-)-and 86Rb(+)-loaded cells with the phosphotyrosine phosphatase inhibitor sodium orthovanadate (200 microM) potentiated isotope efflux triggered by mild hypotonicity (10-20%) but did not further increase the efflux in response to more vigorous osmotic stimulation (30% hypotonicity). The tyrosine kinase inhibitors herbimycin A and genistein largely reduced the osmoshock-induced efflux in both control and vanadate-pretreated cells, while not affecting calcium-activated 86Rb+ efflux. Potentiation of the RVD response by vanadate was confirmed by direct measurements of hypotonicity-induced changes in cell volume. Hypotonic shock alone triggered a rapid and transient increase in tyrosine phosphorylation of several proteins as well as phosphorylation of mitogen-activated protein kinase. Furthermore, the potentiating effects of vanadate on hypotonicity-induced ion efflux and mitogen-activated protein (MAP) kinase phosphorylation were mimicked by epidermal growth factor. Neither vanadate nor epidermal growth factor provoked a RVD-like ionic response under isotonic conditions. These results indicate that tyrosine phosphorylation is an essential step in the RVD response and suggest a novel role of growth factors in the cellular defense against osmotic stress.

Benzoquinones↗

PubMed1993

Heat-stable enterotoxin activation of immunopurified guanylyl cyclase C. Modulation by adenine nucleotides.

We studied the activation and inactivation of recombinant guanylyl cyclase (GC) C stably expressed in human kidney 293 cells. Activation of GC-C by heat-stable enterotoxin (STa), Cd2+, hemin, or the detergent Triton X-100 was followed by a rapid inactivation of the enzyme. Adenine nucleotides were found to prevent the inactivation process in native membranes, as well as following enzyme solubilization and immunopurification. Inactivation of GC-C was not associated with dephosphorylation. However, the phosphorylation of GC-C was promoted by phorbol esters, known activators of protein kinase C. The activation of purified GC-C by STa required the presence of a nonspecific factor as exemplified by bovine serum albumin. When immunopurified GC-C, stabilized by ATP and bovine serum albumin, was analyzed by SDS-polyacrylamide gel electrophoresis under nonreducing conditions, proteins with almost twice the molecular mass (220 and 245 kDa) of the monomer were observed. The mobility of these high M(r) GC-C forms was reduced by STa, suggesting that STa induces a conformation change in a preexisting GC-C dimer. These results indicate that ATP interacts directly with GC-C, stabilizing its active conformation and that the activation of GC-C may occur in the absence of other specific regulatory factors.

Adenosine Triphosphate↗

PubMed1993

Calcium ionophore plus excision induce a large conductance chloride channel in membrane patches of human colon carcinoma cells HT-29cl.19A.

In excised inside-out membrane patches of the human colon carcinoma HT-29cl.19A cells a large conductance (373 +/- 10 pS) chloride channel was found. Channel activity could only be observed after excision of patches from cells incubated with calcium ionophore. The channel was never observed in cell-attached patches. The channel was strongly voltage dependent, being open only between +30 and -30 mV clamp potentials. The selectivity sequence among anions, deduced from reversal potentials, was I > Br > Cl > F > gluconate. The PNa/PCl was 0.09. Although a similar type of channel has been described earlier, this is the first report stating its appearance in patches of intestinal epithelial cells requiring the combined action of Ca2+ ionophore and excision, suggesting its control by an intracellular compound.

Calcimycin↗

PubMed1993

Regulation of chloride channels in the human colon carcinoma cell line HT29.cl19A.

Chloride (Cl-) channels are important in the regulation of salt and water transport in secretory epithelial cells. A disturbed Cl- secretion is the most consistent characteristic in the genetic disease cystic fibrosis. An outwardly rectifying Cl- channel (OR) with a conductance of 25-50 pS had been proposed to play a major role in Cl- secretion. Activation by Ca2+ and the protein kinases (PK) A and C (at less than 10 nM Ca2+) as well as inhibition by PKC (at 1 microM Ca2+) has been reported. In the present study, we have identified and characterized the OR in HT29.cl19A human colon carcinoma cells. The OR displayed a conductance of 31 +/- 4 pS (n = 25). Its open probability in 10 nM Ca2+ was voltage-dependent in 50% of the patches, starting from 0.2 at -70 mV to 0.8 at 70 mV. The spontaneous activation in excised inside-out patches at -60 mV was Ca(2+)-dependent and decreased from 29% in 1 mM Ca2+ to 2% in 10 nM Ca2+. Active OR were found in (a) 25% of patches exposed to 10 nM Ca2+, ATP and cAMP only, (b) 42% of the patches exposed to 10 nM Ca2+, ATP and the catalytic subunit of PKA (CAK) and (c) 67% of the patches exposed to 1 mM Ca2+, ATP plus CAK. Inhibition of voltage-activated channels by addition of PKC in 1 microM or 1 mM Ca2+ was not observed. Attempts to activate the OR in cell-attached patches by increasing cAMP levels under different experimental conditions were unsuccessful.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1993

The value of in vivo electrophysiological measurements for monitoring functional adaptation after massive small bowel resection in the rat.

The process of functional adaptation after extensive small bowel resection is complex and imprecisely understood. In vivo electrophysiological measurements for monitoring the functional adaptive process after massive small bowel resection in Brown-Norway rats were evaluated. Rats underwent either a sham operation (SH) or a 90% small bowel resection (SB). Standard rat chow was fed in unlimited quantities. At three or 10 weeks after operation, jejunal and ileal transepithelial potential differences (PD, mV) were determined. Electrogenic ion transport in the villus was measured after glucose (sodium coupled active glucose absorption; PD-glu) and in the crypt, after theophylline infusion (theophylline stimulated chloride secretion; PD-theo). Biopsies were taken simultaneously. Each experimental group consisted of three to five animals. At three weeks the PD-theo and PD-glu in SB rats were significantly lower than in SH rats in both jejunal and ileal segments. At 10 weeks PD-theo and PD-glu were significantly diminished in the jejunal segment of the SB rats compared with the SH rats. The values of PD-theo and PD-glu in the ileal segments were, however, no longer different between the two groups. Three and 10 weeks after operation the length of the villi in the SB group was increased significantly compared with the SH controls. These results indicate that in the early phase of adaptation in vivo electrophysiological variables do not correlate with histological changes in the SB rats. This might be due to cell immaturity resulting from an increased rate of cell turnover or lack of intercellular tight junctions. This hypothesis is supported by a recovery of PD responses in the ileum 10 weeks after resection.

Adaptation, Physiological↗

PubMed1992

Nucleotide-activated chloride channels in lysosomal membranes.

Lysosomal membrane vesicles purified from rat liver contain a basal chloride conductance that was enhanced in the presence of ATP, non-hydrolysable ATP-analogs and, to a lesser extent, GTP. Other nucleotides, including AMP, ADP and cAMP, as well as CTP and UTP were not effective. Following fusion of the vesicles with an artificial phosphatidylethanolamine/phosphatidylserine bilayer, we found that ATP gamma S dramatically increased the incidence of 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS)-sensitive chloride channels with a unitary slope conductance of approx. 40 pS in 300 mM/50 mM KCl buffers and 120 pS in symmetrical 300 mM KCl buffers. Since similar results were obtained with AMP-PNP, the results indicate that lysosomes contain a chloride permeable ion channel that is activated by ATP through allosteric interaction.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1992

Dual role for protein kinase C alpha as a regulator of ion secretion in the HT29cl.19A human colonic cell line.

The involvement of protein kinase C (PKC) in the regulation of intestinal ion secretion was studied in polarized monolayers of the HT29cl.19A human colon carcinoma cell line. Carbachol, phorbol esters [PMA (phorbol 12-myristate 13-acetate) and PDB (phorbol 12,13-dibutyrate)] and 8-bromo cyclic AMP (8-Br-cAMP) induced Cl secretion, as measured by a rise in the short-circuit current (ISC). The electrical response to carbachol coincided with a transient translocation of PKC alpha from the soluble to the particulate fraction. The carbachol-, PDB- and 8-Br-cAMP-induced ISC responses were inhibited by pretreatment of the cells with PMA (0.5 microM) for 2 h, a time period in which PKC alpha, beta 1 and gamma levels were not changed. As shown by 86Rb+ and 125I- efflux studies, the main targets for this inhibition were basolateral K+ transporters rather than apical Cl- channels. Prolonged exposure to PMA (24 h) led to a 60% recovery of the 8-Br-cAMP response, but not of the carbachol- or PDB-provoked secretion. As shown by immunoblotting with PKC-isoenzyme-specific antisera, the recovery of the 8-Br-cAMP response coincided with the down-regulation of PKC alpha, whereas the levels of PKC beta 1 and gamma were unmodified. These results suggest that PKC alpha, but not PKC beta 1 or gamma, is involved in both acute stimulation and chronic inhibition of ion secretion in the HT29cl.19A colonic cell line.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1992

Regulation of chloride transport in cultured normal and cystic fibrosis keratinocytes.

Cultured normal (N) cystic fibrosis (CF) keratinocytes were evaluated for their Cl(-)-transport properties by patch-clamp-, Ussing chamber- and isotopic efflux-measurements. Special attention was paid to a 32 pS outwardly rectifying Cl- channel which has been reported to be activated upon activation of cAMP-dependent pathways in N, but not in CF cells. This depolarization-induced Cl- channel was found with a similar incidence in N and CF apical keratinocyte membranes. However, activation of this channel in excised patches by protein kinase (PK)-A or PK-C was not successful in either N or CF keratinocytes. Forskolin was not able to activate Cl- channels in N and CF cell-attached patches. The Ca(2+)-ionophore A23187 activated in cell-attached patches a linear 17 pS Cl- channel in both N and CF cells. This channel inactivated upon excision. No relationship between the cell-attached 17 pS and the excised 32 pS channel could be demonstrated. Returning to the measurement of Cl- transport at the macroscopic level, we found that a drastic rise in intracellular cAMP induced by forskolin did in N as well as CF cells not result in a change in the short-circuit current (Isc) or the fractional efflux rates of 36Cl- and 125I-. In contrast, addition of A23187 resulted in an increase of the Isc and in the isotopic anion efflux rates in N and CF cells. We conclude that Cl(-)-transport in cultured human keratinocytes can be activated by Ca2+, but not by cAMP-dependent pathways.

Biological Transport↗

PubMed1992

Biphasic increase of apical Cl- conductance by muscarinic stimulation of HT-29cl.19A human colon carcinoma cell line: evidence for activation of different Cl- conductances by carbachol and forskolin.

The modulation of ion transport pathways in filter-grown monolayers of the Cl(-)-secreting subclone (19A) of the human colon carcinoma cell line HT-29 by muscarinic stimulation was studied by combined Ussing chamber and microimpalement experiments. Basolateral addition of 10(-4) M carbachol induced a complex poly-phasic change of the cell potential consisting of (i) a fast and short (30-sec) depolarization of 15 +/- 1 mV from a resting value of -52 +/- 1 mV and an increase of the fractional resistance of the apical membrane (first phase), (ii) a repolarization of 22 +/- 1 mV leading to a hyperpolarization of the cell (second phase), (iii) a depolarization of 11 +/- 1 mV and a decrease of the fractional resistance of the apical membrane (the third phase), (iv) and sometimes, a hyperpolarization of 6 +/- 1 mV and an increase of the fractional resistance of the apical membrane (fourth phase). The transepithelial potential increased with a peak value of 2.4 +/- 0.3 mV (basolateral side positive). The transepithelial PD started to increase (serosa positive), coinciding with the start of the second phase of the intracellular potential change, and continued to increase during the third phase. Ion replacements and electrical circuit analyses indicate that the first phase is caused by increase of the Cl- conductance in the apical and basolateral membrane, the second phase by increased K+ conductance of the basolateral membrane, and the third phase and the fourth phase by increase and decrease, respectively, of an apical Cl- conductance. The first and second phase of the carbachol effect could be elicited also by ionomycin. They were strongly reduced by EGTA. Phorbol dibutyrate (PDB) induced a sustained depolarization of the cell and a decrease of the apical fractional resistance. The results suggest that two different types of Cl- channels are involved in the carbachol response: one Ca2+ dependent and a second which may be PKC sensitive. In the presence of a supramaximal concentration of forskolin, carbachol evoked a further increase of the apical Cl- conductance. It is concluded that the short-lasting carbachol/Ca(2+)-dependent Cl- conductance is different from the forskolin-activated conductance. The increase of the Cl- conductance in the presence of forskolin by carbachol may be due to activation of different Cl- channels or to modulation of the PKA-activated Cl- channels by activated PKC.

Calcium↗

PubMed1992

Regulation of apical Cl- conductance and basolateral K+ conductances by phorbol esters in HT-29cl.19A cells.

The effect of phorbol esters [4 beta-phorbol 12,13-dibutyrate (PDB) and phorbol 12-myristate 13-acetate (PMA)] on potential differences and resistances was studied with the conventional microelectrode technique applied to confluent filter-grown monolayers of the human colon carcinoma cell line HT-29cl.19A. Phorbol esters (PDB or PMA from 10(-7) to 10(-6) M) evoked 1) a transient increase in the transepithelial potential difference (peak value 3.5 +/- 0.5 mV), 2) a depolarization of the cell potential by 23 +/- 2 mV at the peak of the transepithelial potential change and a continued decrease during the decline of the transepithelial potential, and 3) a decrease of the fractional resistance of the apical membrane consisting of two phases, a relative rapid one (time constant 1.2 +/- 0.2 min) and a much slower further decrease during the second phase (time constant 34 +/- 1 min). Ion replacements and electrical circuit analyses indicate that PDB activates an apical Cl- conductance and slowly inhibits the basal K+ conductance of the basolateral membrane. PDB reduced the transepithelial response to forskolin due to inhibition of the basal K+ conductance. The Ca2+ ionophore ionomycin accelerated that effect of PDB. Staurosporine inhibited the effects of PDB, suggesting that the PDB effects are mediated via activation of a protein kinase C.

Calcium↗