A. Vaandrager
Utrecht University, Biochemistry And Cell Biology, Faculty Member
Research Interests: Chemistry, Biology, Biological Chemistry, Gene expression, Biological Sciences, and 15 moreDNA, Cell line, Humans, Bacterial Toxins, Enterotoxins, Animals, Glycosylation, CHEMICAL SCIENCES, Intestines, Amino Acid Sequence, Drug Stability, Guanylate Cyclase, cyclic GMP, Adenosine Triphosphate, and Enterotoxin
The potential role of polyphosphoinositide (PPI) metabolism as a signal-transduction mechanism in apical membranes of polarized epithelial cells was evaluated by examining the formation and breakdown of PPI in rat intestinal brush-border... more
The potential role of polyphosphoinositide (PPI) metabolism as a signal-transduction mechanism in apical membranes of polarized epithelial cells was evaluated by examining the formation and breakdown of PPI in rat intestinal brush-border membranes (BBM) prelabeled by intraluminal injection of [3H]inositol in vivo or by [gamma-32P]ATP in vitro. Freshly isolated BBM prelabeled with [3H]inositol contained higher amounts of [3H]phosphatidylinositol 4,5-diphosphate compared with a basolateral membrane (BLM) preparation (approximately 14 and 6.8% of total [3H]PPIs, respectively) and were enriched in inositol lipid kinases, diacylglycerol (DAG) kinase, and phosphomonoesterases degrading PPI, inositol bisphosphate, and inositol triphosphate (IP3). In the presence of nonhydrolysable GTP analogues and low Ca2+ (pCa 6-8) or at high Ca2+ alone (pCa 4) endogenous pools of PPI were rapidly depleted by an intrinsic PPI-specific phospholipase C apparently coupled to a GTP-binding protein (G protein...
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The mechanism of adenosine 3',5'-cyclic monophosphate (cAMP)- and Ca(2+)-induced Cl- secretion was studied in monolayers of the colon carcinoma cell line HT-29.cl19A by combined short-circuit current (Isc) and 125I- or 36Cl-... more
The mechanism of adenosine 3',5'-cyclic monophosphate (cAMP)- and Ca(2+)-induced Cl- secretion was studied in monolayers of the colon carcinoma cell line HT-29.cl19A by combined short-circuit current (Isc) and 125I- or 36Cl- efflux measurements. Forskolin, a specific adenylate cyclase activator, was found to induce a large increase in Isc and a two- to threefold increase in 36Cl- efflux solely across the apical border. The fractional efflux of 36Cl-compared with 125I- (basal ratio 1.71 +/- 0.28) did not change significantly in the presence of forskolin (1.91 +/- 0.45). In contrast, the Ca2+ ionophore A23187 did not appreciably affect the Isc but enhanced 36Cl- and 125I- efflux at the apical and basolateral side of the monolayer. Furthermore, the fractional efflux ratio of 36Cl- to 125I- changed dramatically to a value of 0.36 +/- 0.14. Both forskolin- and A23187-induced 36Cl- or 125I- efflux were only weakly inhibited by the putative Cl- channel blocker 5-nitro-2-(3-phenylpr...
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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... more
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 a...
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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... more
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...
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Research Interests: Microbiology, Gastroenterology, Biology, Immunohistochemistry, Medicine, and 15 moreBacterial Toxins, Escherichia coli, Enterotoxins, Animals, Male, Clinical Sciences, Intestines, Rats, Neurosciences, cyclic GMP, Atrial Natriuretic Factor, Intracellular, Enterotoxin, Chlorides, and Paediatrics and reproductive medicine
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... more
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...
Research Interests: Catalysis, Biology, Membrane Proteins, Medicine, Western blotting, and 15 moreBiological Sciences, Animals, Biochemical, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, CHEMICAL SCIENCES, Rats, Autoradiography, G protein, Amino Acid Sequence, Gtp Binding Proteins, Small Intestine, Molecular Sequence Data, microvilli, Medical and Health Sciences, and Cholera toxin
The absorption of electrolytes and water by mammalian small intestine and proximal colon is primarily controlled by an e1ectroneutra1 entry mechanism for Na+ and Cl− in the brush-border membrane of the enterocytes, consisting of parallel... more
The absorption of electrolytes and water by mammalian small intestine and proximal colon is primarily controlled by an e1ectroneutra1 entry mechanism for Na+ and Cl− in the brush-border membrane of the enterocytes, consisting of parallel Na+/H+ and Cl−/HCO3 − exchangers coupled by circular proton movements. Enterotoxins produced by non-invasive micro-organisms (such as choleratoxin, heat-stable and heat-labile Escherichia coli toxin) and neurohumoral factors (such as acetylcholine, vasoactive intestinal peptide[VIP], prostaglandins, bradykinin) promote salt and water secretion by a dual action involving (1) blockade of the antiporters in the villus compartment and (2) Cl− channel opening in the apical membrane of intestinal crypt cells, allowing CT exit down an electrochemical gradient created by a basolateral Na-K-Cl2 entry process. During Cl− secretion, the accumulation of Na+ and K+ in the enterocyte is prevented by Na+ exit through the Na+/K+-pump and K+ exit through secretagogue-sensitive K+ channels in the basolateral membrane.
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Publisher Summary Cyclic guanosine monophosphate (cGMP) was established as an important regulator of intestinal ion transport in the late seventies, when it was shown to be the intracellular mediator of salt and water secretion induced by... more
Publisher Summary Cyclic guanosine monophosphate (cGMP) was established as an important regulator of intestinal ion transport in the late seventies, when it was shown to be the intracellular mediator of salt and water secretion induced by Escherichia coli heat-stable enterotoxin (STa). The recent finding of a distinctive endogenous activator of intestinal guanylyl cyclase (guanylin) affirms that the intestine possesses a unique cGMP-signaling pathway for the physiological regulation of ion transport. The regulation of fluid secretion by cGMP, however, has been thought to be restricted to the intestine. However, the recent detection of some of the components of the “intestinal cGMP pathway” (GC-C and guanylin) in other tissues suggests that it is expressed more generally than previously assumed. The chapter discusses the role of cGMP pathway in the intestine as a regulator of transepithelial ion and fluid transport.
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A recently cloned isoform of cGMP-dependent protein kinase (cGK), designated type II, was implicated as the mediator of cGMP-provoked intestinal Cl − secretion based on its localization in the apical membrane of enterocytes and on its... more
A recently cloned isoform of cGMP-dependent protein kinase (cGK), designated type II, was implicated as the mediator of cGMP-provoked intestinal Cl − secretion based on its localization in the apical membrane of enterocytes and on its capacity to activate cystic fibrosis transmembrane conductance regulator (CFTR) Cl − channels. In contrast, the soluble type I cGK was unable to activate CFTR in intact cells, although both cGK I and cGK II could phosphorylate CFTR in vitro . To investigate the molecular basis for the cGK II isotype specificity of CFTR channel gating, we expressed cGK II or cGK I mutants possessing different membrane binding properties by using adenoviral vectors in a CFTR-transfected intestinal cell line, and we examined the ability of cGMP to phosphorylate and activate the Cl − channel. Mutation of the cGK II N-terminal myristoylation site (Gly 2 → Ala) reduced cGK II membrane binding and severely impaired cGK II activation of CFTR. Conversely, a chimeric protein, in...
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Atrial natriuretic peptide (ANP) and nitric oxide (NO) are key regulators of ion and water transport in the kidney. Here, we report that these cGMP-elevating hormones stimulate Ca 2+ reabsorption via a novel mechanism specifically... more
Atrial natriuretic peptide (ANP) and nitric oxide (NO) are key regulators of ion and water transport in the kidney. Here, we report that these cGMP-elevating hormones stimulate Ca 2+ reabsorption via a novel mechanism specifically involving type II cGMP-dependent protein kinase (cGK II). ANP and the NO donor, sodium nitroprusside (SNP), markedly increased Ca 2+ uptake in freshly immunodissected rabbit connecting tubules (CNT) and cortical collecting ducts (CCD). Although readily increasing cGMP, ANP and SNP did not affect Ca 2+ and Na + reabsorption in primary cultures of these segments. Immunoblot analysis demonstrated that cGK II, and not cGK I, was present in freshly isolated CNT and CCD but underwent a complete down-regulation during the primary cell culture. However, upon adenoviral reexpression of cGK II in primary cultures, ANP, SNP, and 8-Br-cGMP readily increased Ca 2+ reabsorption. In contrast, no cGMP-dependent effect on electrogenic Na + transport was observed. The membr...
Research Interests: Chemistry, Kinetics, Biology, Calcium, Medicine, and 15 moreMultidisciplinary, Cell Culture, Humans, Internal Medicine, Animals, Rats, Protein Kinase, Primary Culture, Rabbits, Recombinant Proteins, Sodium Nitroprusside, Atrial Natriuretic Factor, Cell Membrane, reverse transcriptase polymerase chain reaction, and Kidney cortex
Research Interests: Biology, Calcium, Cell Biology, Medicine, Ion Channels, and 15 moreMammals, Humans, Platelet aggregation, Nitric oxide, Animals, Homeostasis, Endothelial cell, Isoenzymes, Muscle contraction, Genetic variation, Protein Kinase, Second Messengers, cyclic GMP, Biochemistry and cell biology, and Atrial Natriuretic Factor
Research Interests: Microbiology, Biophysics, Chemistry, Cystic Fibrosis, Medical Microbiology, and 15 moreMembrane Biology, Calcium, Membrane Proteins, Medicine, Cell line, Humans, cyclic AMP, Ion Transport, Membrane Potential, Colon Carcinoma, Phorbol ester, Biochemistry and cell biology, Chlorides, Depolarization, and Colonic Neoplasms
Influenza A virus (IAV) infections are a major cause of respiratory disease of humans and animals. Pigs can serve as important intermediate hosts for transmission of avian IAV strains to humans, and for the generation of reassortant... more
Influenza A virus (IAV) infections are a major cause of respiratory disease of humans and animals. Pigs can serve as important intermediate hosts for transmission of avian IAV strains to humans, and for the generation of reassortant strains; this may result in the appearance of new pandemic IAV strains in humans. We have studied the role of the porcine lung collectins surfactant proteins D and A (pSP-D and pSP-A), two important components of the innate immune response against IAV. Hemagglutination inhibition assays revealed that both pSP-D and pSP-A display substantially greater inhibitory activity against IAV strains isolated from human, swine, and horse, than lung collectins from other animal species. The more potent activity of pSP-D results from interactions mediated by the asparagine-linked oligosaccharide located in the carbohydrate recognition domain of pSP-D, which is absent in SP-Ds from other species characterized to date. Presence of this sialylated oligosaccharide moiety...
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Research Interests: Genetics, Chemistry, Cystic Fibrosis, Biology, Biological Chemistry, and 15 moreMedicine, Biological Sciences, Cell line, Animals, Gene transfer techniques, Biological, Phosphorylation, CHEMICAL SCIENCES, Rats, Isoenzymes, Chloride Channel, Protein Kinase, cyclic GMP, Gene Transfer, and Medical and Health Sciences
Research Interests: Biology, Cell Biology, Biological Chemistry, Medicine, Biological Sciences, and 15 moreCell line, Humans, Animals, Biological, Lipid metabolism, CHEMICAL SCIENCES, Isoenzymes, Chloride Channel, Enzyme activity, Base Sequence, Ion Transport, cyclic GMP, Molecular Sequence Data, Cell Membrane, and Medical and Health Sciences
Research Interests: Chemistry, Biology, Enzyme Inhibitors, Biological Chemistry, Comparative Study, and 15 moreBiological Sciences, Cell line, Animals, Biological, Conductance, Brush Border, Enzyme, CHEMICAL SCIENCES, Cattle, Intestines, Chloride Channel, Amino Acid Profile, cyclic GMP, Adenosine Triphosphate, and Cell Membrane
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ABSTRACTCryptococcus neoformansis an opportunistic pathogen invading the immunocompromised host. Infection starts with the inhalation of acapsular or sparsely encapsulated cells, after which capsule synthesis is initiated. The capsule is... more
ABSTRACTCryptococcus neoformansis an opportunistic pathogen invading the immunocompromised host. Infection starts with the inhalation of acapsular or sparsely encapsulated cells, after which capsule synthesis is initiated. The capsule is the main virulence factor of this yeast-like fungus. Pulmonary surfactant protein D (SP-D) is an important component of the local innate defense system. In the present study, interactions of SP-D with intactC. neoformanscells and their isolated capsular components were investigated. Although encapsulated cryptococci were bound, SP-D showed the highest affinity for acapsularC. neoformans. Only acapsular cryptococci were aggregated by SP-D. Furthermore, the cryptococcal capsular components glucuronoxylomannan (GXM) and mannoprotein 1 (MP1) were bound with relatively high affinity, in contrast to GalXM and MP2. Binding as well as aggregation of acapsularC. neoformansby SP-D could be inhibited by GXM in concentrations that are likely to be present in th...
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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... more
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 phosphorylat...
Research Interests: RNA, Biology, Microcirculation, Immunohistochemistry, Medicine, and 15 moreThrombin, Humans, Internal Medicine, Calcium channels, Phosphorylation, Clinical Sciences, Hemostasis, Autoradiography, Circulation, Aorta, Cytoplasm, cyclic GMP, Cell Adhesion Molecules, Platelet Aggregation Inhibitors, and Cardiovascular medicine and haematology
Research Interests: Biology, Medicine, Oogenesis, Biological Sciences, Fatty acids, and 15 moreFemale, Animals, Blastocyst, Lipid metabolism, Cattle, Embryonic Development, Fatty Acid, Fertilization in Vitro, Oleic Acid, Oocytes, Follicular Fluid, Oocyte, Biology Reproduction, Medical and Health Sciences, and Cumulus Cells
Research Interests: Biology, Medicine, Biological Sciences, Adipose tissue, Animals, and 15 moreIn Vitro Fertilization, Cattle, Oocyte Maturation, Fatty Acid, Oleic Acid, In Vitro Studies, Free Fatty Acid, Stearic Acid, Oocytes, Follicular Fluid, Palmitic Acid, Oocyte, Biology Reproduction, Adverse effect, and Medical and Health Sciences
Research Interests: Biochemistry, Chemistry, Biology, Medicine, Humans, and 15 moreMutation, Animals, Endoplasmic Reticulum, Lysine, Arginine, Cysteine, Amino Acid Sequence, Base Sequence, Golgi Apparatus, Palmitic Acid, Molecular Sequence Data, CHO cells, Cricetinae, Chinese hamster ovary cells, and Cardiovascular medicine and haematology
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Nitrovasodilators, such as sodium nitroprusside (SNP), release nitric oxide (NO) and stimulate intestinal electrolyte transport. However, the second messengers involved in this process are unknown. NO stimulates soluble guanylate cyclase... more
Nitrovasodilators, such as sodium nitroprusside (SNP), release nitric oxide (NO) and stimulate intestinal electrolyte transport. However, the second messengers involved in this process are unknown. NO stimulates soluble guanylate cyclase activity in other tissues, but stimulation of this enzyme has not previously been described for intestine. We report a 20-fold increase in guanosine 3',5'-cyclic monophosphate (cGMP) production by radioimmunoassay in colonic mucosal strips stimulated with SNP. SNP also caused a significant increase in prostaglandin (PG) E2 release but did not stimulate release of the prostanoids thromboxane B2 or 6-keto-PGF1alpha. Stimulation of isolated colonic crypts and the remaining subepithelial mucosa demonstrated that the latter was the major source of the increases in cGMP and PGE2. Immunostaining of colonic mucosa revealed minimal basal cGMP immunoreactivity but large increases in abundance, localizing to the subepithelium, after SNP treatment. Unde...
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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... more
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 h...
Research Interests: Thermodynamics, Kinetics, Biological Chemistry, Biological Sciences, Cell line, and 13 moreHumans, Bacterial Toxins, Enterotoxins, Biological, Phosphorylation, Heme, CHEMICAL SCIENCES, Isoenzymes, Transfection, Bovine Serum Albumin, Recombinant Proteins, Guanylate Cyclase, and Adenosine Triphosphate
The mutant Chinese hamster ovary cell line MT58 contains a thermosensitive mutation in CTP:phosphocholine cytidylyltransferase, the regulatory enzyme in the CDP-choline pathway. As a result, MT58 cells have a 50% decrease in their... more
The mutant Chinese hamster ovary cell line MT58 contains a thermosensitive mutation in CTP:phosphocholine cytidylyltransferase, the regulatory enzyme in the CDP-choline pathway. As a result, MT58 cells have a 50% decrease in their phosphatidylcholine (PC) level within 24 h when cultured at the nonpermissive temperature (40 degrees C). This is due to a relative rapid breakdown of PC that is not compensated for by the inhibition of de novo PC synthesis. Despite this drastic decrease in cellular PC content, cells are viable and can proliferate by addition of lysophosphatidylcholine. By [(3)H]oleate labeling, we found that the FA moiety of the degraded PC is recovered in triacylglycerol. In accordance with this finding, an accumulation of lipid droplets is seen in MT58 cells. Analysis of PC-depleted MT58 cells by electron and fluorescence microscopy revealed a partial dilation of the rough endoplasmic reticulum, resulting in spherical structures on both sites of the nucleus, whereas the morphology of the plasma membrane, mitochondria, and Golgi complex was unaffected. In contrast to these morphological observations, protein transport from the ER remains intact. Surprisingly, protein transport at the level of the Golgi complex is impaired. Our data suggest that the transport processes at the Golgi complex are regulated by distal changes in lipid metabolism.
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Research Interests: Endocrinology, Immunohistochemistry, Enzyme Inhibitors, Apoptosis, Animal Production, and 17 moreCell Division, Caspases, Cell line, Humans, cyclic AMP, Female, Clinical Sciences, Caspase, Epithelial cells, Veterinary Sciences, Cell Proliferation, Ovary, Luteinizing Hormone, Cell Survival, Tumor Growth, Ligands, and Ovarian Surface Epithelium
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A large percentage (up to 70%) of 36Cl- influx in brush-border membrane vesicles from rat small intestine under equilibrium exchange conditions was found to be mediated by SITS-inhibitable anion exchange. This Cl-/anion exchange could be... more
A large percentage (up to 70%) of 36Cl- influx in brush-border membrane vesicles from rat small intestine under equilibrium exchange conditions was found to be mediated by SITS-inhibitable anion exchange. This Cl-/anion exchange could be measured 10-15-times more sensitive by determining the uptake of trace amounts of 125I- driven by a large Cl- gradient (in greater than out) generated by passing the vesicles through an anion-exchange column. Voltage clamping of the vesicle membrane with K+ and valinomycin did not effect the chloride driven 125I- uptake, showing that the 'overshooting' I- uptake was not mediated by an electrical diffusion potential, as might be generated by the Cl- gradient in the presence of a chloride channel. The Cl-/anion exchange was further characterized in brush-border membrane vesicles from both rat ileum and jejunum by studying the inhibitory action of various anions on the Cl- driven I- uptake. NO3-, Cl-, SCN- and formate at 2 mM could inhibit Cl-/I- exchange for more than 80%. The ileal brush-border membrane vesicles displayed a clear heterogeneity with respect to the inhibitory action of SO2-(4), SITS and HCO-3 on Cl-/I- exchange. Approximately 30% of the Cl-/I- exchange was insensitive to SO2-(4) and showed a relatively low sensitivity to SITS (IC50 = 1 mM) but could be inhibited for 80% by 2 mM HCO-3. Presumably this component represents Cl-/OH- or Cl-/HCO-3 exchange. The residual 70% showed a high sensitivity to SO2-(4) (IC50 = 0.5 mM) and SITS (IC50 = 2.5 microM) but was less sensitive to HCO-3. This part of the exchange activity showed inhibition characteristics very similar to the Cl-/I- exchange in the jejunal vesicles. The latter process was also inhibited for 80% by 2 mM oxalate. As discussed in this paper both exchangers may be involved in the electroneutral transport of NaCl across the apical membrane of the small intestinal villus cell.
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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... more
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.