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J Bijman

Publications and source records attributed to J Bijman.

At least 37 records · Page 2Linked to original sources
PubMed1993

Low-conductance chloride channels in IEC-6 and CF nasal cells expressing CFTR.

The properties of the cystic fibrosis gene product (CFTR) were studied by expression of cloned cDNA in different cell systems. Infection of both simian fibroblast (Vero) cells and immortalized CF nasal polyp cells (NCF3A) with a vaccinia virus encoding CFTR induced forskolin-induced Cl- permeability and low-conductance (8 pS) Cl- channels. By stable transfection of the rat intestinal crypt-derived cell line IEC-6 we have isolated a clone, IEC-CF7, which expresses CFTR mRNA and antigen. IEC-CF7 cells, but not IEC-6, display forskolin-induced Cl- permeability and multiple linear low-conductance (+/- 8 pS) Cl- channels in cell-attached membrane patches. In excised patches of IEC-CF7 cells, low-conductance Cl- channels could be activated by addition of the catalytic subunit of the adenosine 3',5'-cyclic monophosphate-dependent protein kinase A (PKA) plus ATP. During bath fluid replacement studies, the activated low-conductance channel remained active in the absence of ATP at room temperature and showed saturation kinetics. Rectifying (32 pS) Cl- channels were not observed in either IEC-6 cells or IEC-CF7 cells, indicating that there is no relation between CFTR expression and the incidence of this channel. Our data strongly support the conclusion that CFTR can act as a low-conductance Cl- channel, gated by PKA. The IEC-6-derived cell line IEC-CF7 may prove to be a useful model in the study of CFTR function because of the absence of 32-pS Cl- channel activity and its potential for differentiation.

Adenosine Triphosphate↗
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

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

X-ray micro-analysis of cultured respiratory epithelial cells from patients with cystic fibrosis.

X-ray micro-analysis was carried out on cultured respiratory cells from polyps removed from individuals with and without cystic fibrosis (CF). In a first set of experiments, proper experimental conditions were established. Washing the cells with 300 mmol l-1 mannitol in distilled water was found to give the best removal of the culture medium. The elemental concentrations stabilized in about 10 min after the start of the preincubation. Intracellular [Na] and [Cl] increased slightly with increasing passage number, whereas intracellular [K] decreased. Under resting conditions there were no significant differences in elemental content between CF and control cells, and there were no indications for abnormally high total [Ca] in CF cells. In normal cells, stimulation with a cAMP-analogue resulted in a decrease of cellular [Cl], whereas in CF cells an increase was measured. Exposure of both normal and CF cells to ouabain resulted in decreased [K] and increased [Na] and [Cl] level. The calcium ionophore A23187 had a similar effect on normal cells but did not affect CF cells markedly. Application of amiloride to the apical side of the cells resulted in a decrease of cellular [Na] in CF cells, whereas [Na] in control cells was not affected. The results correspond with what is known about the defective cAMP-regulated transepithelial Cl-transport in CF cells. The effect of the calcium ionophore on cellular electrolyte content is more complicated and may be the result of two separate effects: efflux of Cl- via a Ca(2+)-dependent mechanism and inhibition of the Na(+)-K(+)-ATPase by intracellular Ca2+ ions causing an influx of Na+ and Cl- ions.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

Cultured epithelial cells from patients with cystic fibrosis have an increased expression of the 14 kDa Ca2(+)-binding protein CFA.

The Cystic Fibrosis antigen (CFA) is a 14 kDa. Ca2(+)-binding protein known to be expressed in cells of myeloid origin during normal cell differentiation. CFA serum levels are elevated in Cystic Fibrosis (CF) patients and heterozygotes. We examined the expression of CFA in different cultured epithelial cells from controls and patients with CF. The steady state level of CFA was in general higher in epithelial cells from CF patients compared to control cells and was found to increase during cell aging. The latter difference could be attributed to an increased rate of CFA synthesis rather than to an impairment of CFA degradation or secretion, as shown by pulse chase experiments.

Blood Proteins↗
PubMed1991

G-proteins mediate intestinal chloride channel activation.

The localization of several GTP-binding regulatory proteins in teh apical membrane of intestinal epithelial cells has prompted us to investigate a possible role for G-proteins as modulators of apical Cl- channels. In membrane vesicles isolated from rat small intestine or human HT29-cl.19A colon carcinoma cells, the entrapment of guanosine 5'-O-(3-thiophosphate (GTP gamma S) led to a large increase in Cl- conductance, as evidenced by an increased 125I- uptake and faster SPQ quenching. The enhancement was observed in the presence, but not in the absence of the K+ ionophore valinomycin, indicating that the increased Cl- permeability is not secondary to the opening of K+ channels. The effect of GTP gamma S was counteracted by guanosine 5'-O-(2-thiophosphate (GDP beta S) and appeared to be independent of cytosolic messengers, including ATP, cAMP, and Ca2+, suggesting that protein phosphorylation and/or phospholipase C activation is not involved. Patch clamp analysis of apical membrane patches of HT29-cl.19A colonocytes revealed a GTP gamma S-activated, inwardly rectifying, anion-selective channel with a unitary conductance of 20 +/- 4 pS. No spontaneous channel openings were observed in the absence of GTP gamma S, while the open time probability (Po) increases dramatically to 0.81 +/- 0.09 upon addition with GTP gamma S. Since the electrophysiological characteristics and regulatory properties of this channel are markedly different from those of the more widely studied cAMP/protein kinase A-operated channel, we propose the existence of a separate Cl(-)-selective ion channel in the apical border of intestinal epithelial cells. Our results suggest an alternative regulatory pathway in transepithelial salt transport and a possible site for anomalous channel regulation as observed in cystic fibrosis patients.

Animals↗
PubMed1991

Chloride transport in the cystic fibrosis enterocyte.

Molecular mechanisms of intestinal chloride channel regulation and potential abnormalities in electrogenic chloride secretion in intestinal epithelium from cystic fibrosis (CF) patients were investigated by a combination of Ussing chamber, vesicle transport and off-cell patch-clamp analysis. Short circuit current (Isc) measurements in normal and CF rectal biopsies provided evidence for i) a defect in the cAMP-provoked activation of chloride secretion and a (hyper)expression of cAMP-dependent potassium secretion in all CF patients examined (n = 11); ii) a defect in the carbachol-provoked chloride secretion and a (hyper)expression of carbachol-induced potassium secretion in 6/11 patients; iii) a residual (but still impaired) carbachol-induced chloride secretion in 5/11 CF patients (including 2 sibs). The latter class of CF patients appeared to consist genetically of compound heterozygotes for the major delta-F508 deletion, suggesting a correlation between the nature of the mutation in the CF gene and the severity of the chloride secretory defect in CF intestine. In our search for a regulatory function of GTP-binding (G-) proteins detected previously in the luminal membrane of rat and human intestinal epithelial cells, evidence was found for the presence of a GTP[S]-activatable- and GDP[S]-inhibitable chloride conductance in the apical membrane of rat enterocytes and human colonocytes. In excised patches of human colonocyt membranes, this G-proteine-sensitive chloride conductance was identified further as a novel type of chloride channel (20pS; inwardly rectifying) that was different from the 33pS outwardly rectifying chloride channel activatable by cAMP-dependent proteinkinase (PK-A) and voltage depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Electrophysiological studies of forskolin-induced changes in ion transport in the human colon carcinoma cell line HT-29 cl.19A: lack of evidence for a cAMP-activated basolateral K+ conductance.

Forskolin (i.e., cAMP)-modulation of ion transport pathways in filter-grown monolayers of the Cl(-)-secreting subclone (19A) of the human colon carcinoma cell line HT29 was studied by combined Ussing chamber and microimpalement experiments. Changes in electrophysiological parameters provoked by serosal addition of 10(-5) M forskolin included: (i) a sustained increase in the transepithelial potential difference (3.9 +/- 0.4 mV), (ii) a transient decrease in transepithelial resistance with 26 +/- 3 omega.cm2 from a mean value of 138 +/- 13 omega.cm2 before forskolin addition, (iii) a depolarization of the cell membrane potential by 24 +/- 1 mV from a resting value of -50 +/- 1 mV and (iv) a decrease in the fractional resistance of the apical membrane from 0.80 +/- 0.02 to 0.22 +/- 0.01. Both, the changes in cell potential and the fractional resistance, persisted for at least 10 min and were dependent on the presence of Cl- in the medium. Subsequent addition of bumetanide (10(-4) M), an inhibitor of Na/K/2Cl cotransport, reduced the transepithelial potential, induced a repolarization of the cell potential and provoked a small increase of the transepithelial resistance and fractional apical resistance. Serosal Ba2+ (1 mM), a known inhibitor of basolateral K+ conductance, strongly reduced the electrical effects of forskolin. No evidence was found for a forskolin (cAMP)-induced modulation of basolateral K+ conductance. The results suggest that forskolin-induced Cl- secretion in the HT-29 cl.19A colonic cell line results mainly from a cAMP-provoked increase in the Cl- conductance of the apical membrane but does not affect K+ or Cl- conductance pathways at the basolateral pole of the cell. The sustained potential changes indicate that the capacity of the basolateral transport mechanism for Cl- and the basal Ba(2+)-sensitive K+ conductance are sufficiently large to maintain the Cl- efflux across the apical membrane. Furthermore, evidence is presented for an anomalous inhibitory action of the putative Cl- channel blockers NPPB and DPC on basolateral conductance rather than apical Cl- conductance.

Biological Transport↗
PubMed1991

Immunological localization of cystic fibrosis candidate gene products.

The recent identification of the cystic fibrosis (CF) gene and its putative protein product, the CF transmembrane conductance regulator (CFTR), enabled us to synthesize oligopeptides corresponding with a predicted extracellular domain (position 103-117; peptide A) and a cytoplasmic domain (position 501-515; peptide B) constituting the phenylalanine deletion (F 508) observed in the majority of CF mutations. Immunobiochemical studies with antibodies directed against these peptides revealed the presence of two CFTR candidate proteins (155 and 195 kDa) in various types of epithelial cells. Immunolocalization studies performed on slices of human duodenum showed the strongest expression in the endoplasmic reticulum (RER) of the mucus-producing Goblet cells. Labeling is also demonstrated in the RER and apical membranes of villus and crypt cells, however, to a weaker extent.

Amino Acid Sequence↗
PubMed1991

Ion transport abnormalities in rectal suction biopsies from children with cystic fibrosis.

Abnormalities in transepithelial electrolyte transport in cystic fibrosis rectum were analyzed by short-circuit current measurements on 11 control subjects and 11 subjects with cystic fibrosis in a modified Ussing chamber. As judged by the amiloride-sensitive component of the short-circuit current, electrogenic sodium absorption appeared unmodified in cystic fibrosis. In contrast, the short-circuit current response to specific stimuli of both cyclic adenosine monophosphate (cAMP)- and calcium-mediated chloride secretion was drastically altered in all of the cystic fibrosis biopsy specimens examined. Stimulation of the cAMP pathway by 8-bromo cAMP or forskolin resulted in a sustained increase in short-circuit current in control tissues (+ 2.51 +/- 0.63 microA/cm2) but in a slight change in the opposite direction in cystic fibrosis (-0.56 +/- 0.49 microA/cm2; P less than 0.05). Carbachol, a calcium-linked secretagogue, provoked a transient increase in short-circuit current in all of the control tissues (peak response, + 26.69 +/- 3.63 microA/cm2) but a transient change in the opposite direction in 6 of 11 cystic fibrosis tissues (-12.46 +/- 4.64 microA/cm2; P less than 0.05). In 2 of 11 patients with cystic fibrosis, however, a significant but subnormal and transient increase in short-circuit current was observed (+ 2.62 +/- 0.04 microA/cm2; P less than 0.05), whereas in 3 of 11 patients with cystic fibrosis a transient change in the opposite direction (-9.83 +/- 2.20 microA/cm2; P less than 0.05) was followed by a small and transient increase (+ 2.89 +/- 0.83 microA/cm2; P less than 0.05). Using the calcium-mediated secretory response therefore, patients with cystic fibrosis could be divided into two categories: a major population showing defective anion secretion but active cation secretion and a subclass (including three siblings) showing residual but subnormal anion secretion. The easy accessibility of rectal samples and the inversed direction of the cAMP- or calcium-provoked short-circuit current is of considerable advantage in the diagnosis of cystic fibrosis.

Adolescent↗
PubMed1991

Value of in vivo electrophysiological measurements to evaluate canine small bowel autotransplants.

This study aimed to develop a non-invasive method for in vivo measurement of the transepithelial potential difference in the canine small bowel and to evaluate this parameter in small bowel autotransplants. In group 0 (control group, n = 4), two intestinal loops were created without disturbing their vascular, neural, and lymphatic supplies. In group I (successful autotransplants, n = 11), two heterotopic small bowel loops were constructed. Long term functional sequelae of vascular, neural, and lymphatic division were studied. Group II (n = 6) consisted of dogs with unsuccessful autotransplants suffering thrombosis of the vascular anastomosis, which resulted in ischaemic small bowel autografts. In group I, values of spontaneous transepithelial potential difference, an index of base line active electrolyte transport, were significantly lower compared with group 0 (p less than 0.05), probably as a result of denervation of the autotransplants. Both theophylline and glucose stimulated potential difference responses, measuring cyclic adenosine monophosphate mediated chloride secretion and sodium coupled glucose absorption respectively, showed negative luminal values in group I at all time points after transplantation. These transepithelial potential difference responses diminished progressively with time. From day 21 onwards both theophylline and glucose stimulated potential difference responses were significantly less than the corresponding responses at day seven (p less than 0.05). Morphometric analysis showed that the reduction of transepithelial potential difference responses preceded degenerative mucosal changes in the heterotopic small bowel autografts. In group II, potential difference responses to theophylline and glucose showed positive luminal values (p<0.01 v group I), probably as a result of passive potassium effusion from necrotic enterocytes.

Animals↗
PubMed1990

The cystic fibrosis defect approached from different angles--new perspectives on the gene, the chloride channel, diagnosis and therapy.

The search for the basic defect in cystic fibrosis (CF) has reached a decisive stage since the recent identification of the responsible gene. Electrophysiological and biochemical research had defined the CF defect as a dysregulation of epithelial chloride channels. The putative protein product of the now identified gene shares properties with other known transport proteins, but it is not necessarily itself a chloride channel protein. Elucidation of the primary cellular defect will certainly have important aetiological and hopefully therapeutic implications. The identification of the major gene mutation already has significant consequences for genetic counselling and prenatal diagnosis. Heterozygote detection at the population level awaits identification of the probably heterogenous mutations on about 30% of the CF chromosomes. At present, about 50% of CF patients are homozygous for the recently identified major CF mutation.

Chlorides↗
PubMed1990

Chloride transport in cultured nasal epithelium of cystic fibrosis patients.

In this study, nasal polyp epithelial cells from control and cystic fibrosis (CF) patients were cultured using a method which allows multiple passages. The cells were tested in Ussing chamber experiments to study transcellular ion transport. Cultured CF nasal polyp cells did not exhibit spontaneous transcellular chloride transport in the presence of amiloride, in contrast to normal cells. Forskolin increased the short circuit current (Isc) in control but not CF cells. Forskolin and isoproterenol increased the cAMP levels in control and CF cells. Histamine, bradykinin and isoproterenol transiently increased the intracellular calcium level and caused a parallel increase of the transcellular chloride current in both normal and CF cells. The transient effects of isoproterenol were not sensitive to the beta blocker atenol and could not be mimicked by forskolin. We conclude that in cultured nasal polyp cells a difference in chloride transport activity between CF and control cells is retained following multiple passages. Our results suggest that the active state of chloride channels in nasal polyp cells does not require activation of a second messenger pathway. This apparently spontaneous activity appears to be reduced in CF cells. The calcium- but not the cAMP-dependent activation of transepithelial chloride secretion is at least partially preserved in cultured CF airway epithelium.

Amiloride↗
PubMed1989

Immortalization of nasal polyp epithelial cells from cystic fibrosis patients.

We have developed immortalized epithelial cystic fibrosis (CF) cell lines by infecting cultured nasal polyp cells with a SV40/Adenol2 hybrid virus. The cell lines obtained are epithelial in nature as shown by cytokeratin production and morphology, although cytokeratins 4 and 13 typical of primary nasal polyp cells are produced at a much reduced rate. Ussing chamber experiments showed that the precrisis CF cell line NCF3 was able to perform trans-cellular chloride transport when activated by agents which elevate intracellular calcium. cAMP agonists had no effect on chloride flux in NCF3 as expected for CF cells. The apical chloride channels found with the patch clamp technique in NCF3 and in the postcrisis cell line NCF3A have a conductance similar to that of chloride channels found earlier in normal and CF epithelial cells. The channels show a delay in the onset of activity in off-cell patches and are not activated by increased cAMP levels in the cell. This indicates that immortalized CF epithelial cells will provide a useful model for the study of cystic fibrosis.

Cell Line↗
PubMed1989

Disturbance of fluid and electrolyte transport in cystic fibrosis epithelia.

Studies of the disease CF suggest that the basic defect is related to impaired electrolyte movement in the epithelia of a variety of organs with exocrine function. The disturbances of electrolyte secretion in the organs classically involved in CF range from 1) a decrease in secretion- or uptake of chloride ions in all the organs studied; 2) an increase in sodium uptake in nasal airway epithelium and 3) a decrease in bicarbonate output of the pancreas. In this review an overview is presented of the expression of the CF defect, the abnormalities of fluid and electrolyte secretion in each CF affected organ are considered in more detail with particular emphasis on the hormonal and neuronal (dys)regulation of iron transport systems in epithelial cells.

Biological Transport↗