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H R de Jonge

Publications and source records attributed to H R de Jonge.

At least 55 records · Page 3Linked to original sources
PubMed1992

Phorbol esters stimulate and inhibit Cl- secretion by different mechanisms in a colonic cell line.

Phorbol 12-myristate 13-acetate (PMA) was found to increase both the short-circuit current (Isc) and the efflux of 125I- or 36Cl- in the colonic epithelial cell line HT-29.cl19A. Neither the PMA-provoked rise in Isc nor the stimulation of 125I- efflux was affected by the cyclooxygenase inhibitor indomethacin. The PMA-induced increase in Cl- efflux was not accompanied by a rise in adenosine 3',5'-cyclic monophosphate (cAMP) levels. A prolonged incubation with PMA (3 h), however, inhibited the PMA- and the cAMP-stimulated Isc by greater than 90%, whereas the cAMP-provoked 125I- and 36Cl- efflux was not inhibited. The long-term PMA treatment was found to inhibit the basal and cAMP-provoked 86Rb+ efflux by 65 +/- 9 and 86 +/- 7%, respectively. A 3-h incubation with PMA also strongly inhibited the Ca2+ ionophore A23187-induced increase in 86Rb+ efflux, whereas the A23187-stimulated 125I- efflux was only marginally inhibited. These data suggest that phorbol esters, presumably by activation of protein kinase C, can provoke Cl- secretion in HT-29.cl19A colonocytes independently of a prostaglandin- or cAMP-mediated pathway. Prolonged exposure to PMA, however, causes an inhibition of net electrogenic Cl- secretion by downregulation of the activity of K+ transporters.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

Asymmetrical distribution of G-proteins among the apical and basolateral membranes of rat enterocytes.

The distribution of the alpha and beta subunits of guanosine-nucleotide-binding proteins (G-proteins) among the apical and basolateral membranes of polarized rat enterocytes was investigated by ADP-ribosylation assays in vitro and immunoblotting with G-protein-subunit-specific antisera. The enterocytes were found to express alpha i2, alpha ji3, alpha s and beta subunits, whereas alpha i1 and alpha o subunits could not be detected. The alpha i2 and alpha i3 subunits were located predominantly in the basolateral membrane, in contrast with the alpha s and beta subunits, which were distributed uniformly among both membranes. Furthermore, 39 kDa and 78 kDa proteins, recognized by anti-alpha i1/2 but not anti-alpha i1 or anti-alpha i3 specific antisera, and resistant to ADP-ribosylation by pertussis toxin, were localized exclusively at the apical border. These Gi-related proteins might represent novel members of the G-protein family. Activation of apical G-proteins by GTP or its analogues failed to release the alpha s, alpha i and beta subunits or the 39 kDa and 78 kDa alpha i-like proteins from the membrane, suggesting a functional role for these proteins in the apical membrane itself. Our recent finding of a guanosine 5'-[gamma-thio]triphosphate-sensitive Cl- conductance in the apical membrane of rat enterocytes suggests that one or more of these G-proteins may act as local regulators of specific apical transport functions.

Adenosine Diphosphate Ribose↗
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

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

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↗
PubMed1990

Cumene hydroperoxide, an agent inducing lipid peroxidation, and 4-hydroxy-2,3-nonenal, a peroxidation product, cause coronary vasodilatation in perfused rat hearts by a cyclic nucleotide independent mechanism.

STUDY OBJECTIVE - The aim of the study was to determine whether cumene hydroperoxide, a substance known to induce lipid peroxidation through free radical action, and 4-hydroxy-2,3-nonenal (4-hydroxynonenal), a major aldehyde formed during lipid peroxidation, induce coronary vasodilatation by changing cyclic nucleotide levels. DESIGN - The study involved Langendorff perfused rat hearts, using different concentrations of cumene hydroperoxide and 4-hydroxynonenal, with sodium nitroprusside for comparison. Coronary flow was measured indirectly as retrograde aortic flow, with constant perfusion pressure. Information about the precise localisation of cyclic guanosine monophosphate (cGMP) in the heart was obtained by immunocytochemistry, using a new cGMP antiserum. EXPERIMENTAL MATERIAL - Hearts were from male Wistar rats, body weight 200-250 g. MEASUREMENTS and RESULTS - Both cumene hydroperoxide and 4-hydroxynonenal caused a dose dependent and reversible increase in coronary flow comparable with sodium nitroprusside. With sodium nitroprusside there was a good correlation between extent of vasodilatation and total heart cGMP concentration. Vasodilatation induced by cumene hydroperoxide or 4-hydroxynonenal was not accompanied by increase in total heart cGMP or cAMP (cyclic adenosine monophosphate) concentration. Isoprenaline was used as a positive control for cAMP. cGMP immunostaining was found in coronary vascular smooth muscle after vasodilatation with sodium nitroprusside, but no immunostaining was found in vascular smooth muscle after vasodilatation with cumene hydroperoxide or 4-hydroxynonenal. CONCLUSIONS - Cumene hydroperoxide and 4-hydroxynonenal can provoke reversible coronary vasodilatation in isolated perfused rat hearts by a cyclic nucleotide independent mechanism.

Aldehydes↗
PubMed1989

Characterization of a proton-driven carrier for sialic acid in the lysosomal membrane. Evidence for a group-specific transport system for acidic monosaccharides.

Highly purified lysosomal membrane vesicles, obtained from rat liver lysosomes, were used to study characteristics of NeuAc transport across the lysosomal membrane. Uptake of [14C]NeuAc was found to be strongly influenced by a pH gradient across the membrane. When a proton gradient (pHin greater than pHout) was generated by impermeable buffers, NeuAc uptake above equilibrium level (overshoot) was observed. The influence of membrane diffusion potentials was ruled out by experiments where K+ and valinomycin were present. The overshoot appeared to be specifically produced by protons, since gradients of other cations (Na+ and K+) did not give stimulation. Proton-driven uptake was saturable (Kt = 0.24 mM) and mediated by a single system, as shown by linearity of the Scatchard plot. Stimulation of transport was also obtained by preincubation of vesicles with MgATP and the effect was blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, but not by the protonophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. Monocarboxylic sugars like glycuronic acids were competitive inhibitors of sialic acid transport. Transstimulation of [14C] NeuAc uptake was observed when vesicles were preloaded either with unlabeled NeuAc or with glucuronic acid. The data demonstrate that lysosomal membrane vesicles from rat liver are a suitable system for kinetic studies of solute transport events. The presence of a proton-driven carrier in the lysosomal membrane specific for sialic acid and other acidic sugars, including glucuronic acid, is shown. The possible physiological significance of these findings for the human lysosomal carrier and the patients with a sialic acid transport defect is discussed.

Amino Acids, Diamino↗
PubMed1989

The molecular basis of chloride channel dysregulation in cystic fibrosis.

The opening and closing of chloride (Cl-) channels in the apical membrane of epithelial cells is regulated by hormones, neurotransmitters and enterotoxins (intestine) acting through a variety of intracellular messengers, including cyclic nucleotides (cAMP, cGMP), calcium (Ca) and diacylglycerol (DAG). The chloride impermeability of epithelial membranes observed in cystic fibrosis (CF) patients does not result from a defect in the Cl- conducting properties of the channel or in channel recruitment but stems either from a defect in a key regulator of the channel, presumably a phosphoprotein, or from the hyperactivation of a channel closing mechanism, presumably a protein phosphatase or a down-regulating protein kinase (i.e. protein kinase C). In vitro phosphorylation of isolated intestinal brush border membranes has revealed the existence of a 25,000 molecular weight proteolipid (p25) acting as cosubstrate for both cGMP- and cAMP-dependent protein kinases and cross-reacting with antibodies directed against the cytoplasmic tail of the band 3 anion exchanger from erythrocytes. The putative role of p25 in Cl- channel regulation and its relationship to an unidentified GTP-binding protein recently implicated in Cl- channel activation is discussed on the basis of a regulatory model indicating potential sites of the CF defect at a molecular level.

Cell Membrane Permeability↗
PubMed1987

gamma-Aminobutyric acid production in small and large intestine of normal and germ-free Wistar rats. Influence of food intake and intestinal flora.

In recent hypotheses concerning the pathogenesis of hepatic encephalopathy, gamma-aminobutyric acid (GABA) is claimed to be produced by the colonic flora, although enzymes necessary to generate GABA have been reported to be present in intestinal mucosa. In this study, using normal and germ-free Wistar rats, we determined GABA levels and amino-grams of arterial blood and of venous effluent from small and large bowel. The data indicate that large and small intestinal mucosa significantly contribute to GABA production. In the fasted state GABA concentrations are greater in the venous effluent of the small bowel than in the venous effluent of the large bowel. Feeding increases the arterioportal differences, and uptake in the small bowel is still significantly higher than in the large bowel. This process is not, or can only be to a minor degree, bacterially mediated, because GABA production in the gut both in the fed and fasted state is of similar magnitude in germ-free and normal animals. gamma-Aminobutyric acid release correlates significantly with glutamine uptake in the small bowel of fasted rats. Only a small fraction of the glutamine taken up is needed to account for GABA release, so that conclusions concerning which amino acids may serve as precursors of GABA cannot be drawn. Further studies are needed to delineate the metabolic pathways leading to GABA synthesis.

Aminobutyrates↗
PubMed1986

Modulation of salt permeabilities of intestinal brush-border membrane vesicles by micromolar levels of internal calcium.

A possible modulation of ion permeabilities of rat intestinal brush-border membrane vesicles by Ca2+, a putative second messenger of salt secretion, was explored by three independent methods: (1) measurements of [3H]glucose accumulation driven by a Na+ gradient; (2) stopped-flow spectrophotometry of salt-induced osmotic swelling; (3) 86Rb+, 22Na+ and 36Cl- flux measurements. Cytoskeleton-deprived membrane vesicles were prepared from isolated brushborders by thiocyanate treatment. Intravescicular Ca2+ levels were varied by preincubating vesicles in Ca-EGTA buffers in the presence of the Ca2+-ionophore A23187. At Ca2+free greater than 10(-5) M, initial Na+-dependent glucose uptake in the presence of a 0.1 M NaSCN gradient (but not in its absence) was inhibited by about 50 per cent as compared to EGTA alone (ED50 approximately equal to 10(-6) M Ca2+). By contrast, initial rates of 22Na+ uptake and reswelling rates of vesicles exposed to a NaSCN gradient were increased at least 2-fold by 10(-5) M Ca2+free. Both observations are compatible with a Ca2+-induced increase of the Na+-permeability of the vesicle membrane. The modulation of ion transport was fully reversible and critically dependent on internal Ca2+, suggesting a localization of Ca2+-sensor sites at the inner surface of the microvillous membrane. As shown by radiotracer and osmotic swelling measurements, micromolar Ca2+ additionally increased the flux rate of K+, Rb+, Cl- and NO-3 but did not change the membrane permeability for small uncharged molecules, including glucose and mannitol. The effect of Ca2+ on ion permeabilities could be blocked by Ba2+ (10(-3) M) or Mg2+ (10(-2) M), but not by amiloride (10(-3) M), apamin (2 X 10(-7) M), trifluoperazine (10(-4) M) or quinine (5 X 10(-4) M). At present it is unclear whether Ca2+ activates a nonselective cation and anion channel or multiple highly selective channels in the vesicle membrane.

Animals↗
PubMed1986

Local changes in fractional saturation of cGMP- and cAMP-receptors in intestinal microvilli in response to cholera toxin and heat-stable Escherichia coli toxin.

Cyclic nucleotide modulation of electrolyte transport across intestinal brushborder membranes is initiated by binding of cGMP and cAMP to high-affinity receptors at the interior of the microvilli. Previously these receptors have been identified by photoaffinity-labelling techniques as regulatory domains of cGMP- and cAMP-dependent protein kinases. In the present study, the receptor concentration in isolated brushborder membrane vesicles and their fractional saturation in absorptive and secretory states of the tissue were estimated. In microvillous membrane vesicles isolated from rat small intestine in the absorptive state, about 10% of the total number of cGMP receptors (25.5 pmol/mg protein) and 40% of all cAMP receptors (28.7 pmol/mg protein) were occupied by endogenous cyclic nucleotides. Luminal exposure of the intestinal segments in vivo to heat-stable Escherichia coli toxin for 3-5 min increased the occupancy of cGMP receptors by about 5-fold without affecting receptor-bound cAMP levels. In contrast, incubation with cholera toxin for 2 h increased the fractional saturation solely of cAMP receptors by 2-fold. Addition of heat-stable E. coli toxin to cholera toxin-pretreated segments, again raising the cGMP levels by 5-fold, did not reduce the amount of receptor-bound cAMP. This finding argues against the concept that increased levels of cAMP during cholera would mimick cGMP effects on ion transport by low-affinity binding to microvillar cGMP receptors. This analysis of local changes in cyclic nucleotide levels at the microvillous level might help to explore the mechanism of action of other secretagogues or antidiarrhoeal agents and to delineate a possible compartmentation of cGMP and cAMP pools within the intestinal mucosa responding differently to external signals.

Animals↗
PubMed1986

Internal exposure of rats to benzidine derived from orally administered benzidine-based dyes after intestinal azo reduction.

The role of the rat intestinal flora in the azo reduction of some benzidine-based dyes was studied in vitro and in vivo. The formation of benzidine was measured after anaerobic incubation of direct black 38, direct blue 6 and direct brown 95 in the presence of caecal bacteria in vitro. Benzidine was absorbed from the intestinal tract much better than the parent compounds. Oral administration of direct black 38 or direct brown 95 to Wistar rats results in the urinary excretion of mutagens. After oral administration of these dyes to germ-free Wistar rats no mutagenicity was observed in urine. The present results show that after oral administration, reduction by the intestinal flora should be considered as the first essential step in the biotoxification of benzidine-based dyes.

Administration, Oral↗
PubMed1985

Mechanisms by which cyclic nucleotides and other intracellular mediators regulate secretion.

The regulation of Na+ and Cl- transport across surface membranes and tight junctions of intestinal epithelium is mediated through at least three intracellular messengers: (i) 3',5'-cyclic AMP, activating two types of cyclic AMP-dependent protein kinase, (ii) 3',5'-cyclic GMP, binding to a unique isoenzyme of cyclic GMP-dependent protein kinase, enriched in the intestinal brush border, and (iii) Ca2+ ions, partially acting through calmodulin and a Ca2+/phospholipid-dependent protein kinase (C kinase). Recent data on the subcellular distribution and molecular properties of the high affinity cyclic nucleotide and Ca2+ receptors, their influence on the phosphorylation state of specific membrane proteins, and the possible role of these target proteins in ion transport regulation, are reviewed. The following aspects are accentuated: (1) the asymmetrical compartmentation of cyclic AMP-dependent protein kinase isoenzymes in the enterocyte and its functional implications; (2) the structure and function of microvillous cyclic GMP-dependent protein kinase; (3) the integration of cyclic AMP and cyclic GMP signals through co-phosphorylation of a 25 000 Mr protein in the intestinal-microvilli; (4) the identification of C kinase in villous and crypt cells; (5) various levels of interaction between cyclic nucleotide and Ca2+ signals; and (6) priority areas for future studies on stimulus-secretion coupling.

Animals↗