42 results on '"Van Kerkhove, Emmy"'
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2. Cadmium and transport of ions and substances across cell membranes and epithelia
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Van Kerkhove, Emmy, Pennemans, Valérie, and Swennen, Quirine
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- 2010
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3. A primary culture of mouse proximal tubular cells, established on collagen-coated membranes
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Terryn, Sara, Jouret, Francois, Vandenabeele, Frank, Smolders, Inge, Moreels, Marjan, Devuyst, Olivier, Steels, Paul, and Van Kerkhove, Emmy
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Kidney tubules -- Research ,Cellular control mechanisms -- Research ,Biological transport, Active -- Research ,Sodium channels -- Research ,Glucose metabolism -- Research ,Cell research ,Biological sciences - Abstract
A simple method is described to establish primary cultures of kidney proximal tubule cells (PTC) on membranes. The permeable membranes represent a unique culture surface, allowing a high degree of differentiation since both apical and basolateral membranes are accessible for medium. Proximal tubule (PT) segments from collagenase-digested mouse renal cortices were grown for 7 days, by which time cells were organized as a confluent monolayer. Electron microscopic evaluation revealed structurally polarized epithelial cells with numerous microvilli, basolateral invaginations, and apical tight junctions. Immuno-blotting for markers of distinct parts of the nephron demonstrated that these primary cultures only expressed PT-specific proteins. Moreover immunodetection of distinct components of the receptor-mediated endocytic pathway and uptake of FITC-albumin indicated that these cells expressed a functional endocytotic apparatus. In addition, primary cultures possessed the PT brush-border enzymes, alkaline phosphatase, and [gamma]-glutamyl-transferase, and a phloridzin-sensitive sodium-dependent glucose transport at their apical side. Electrophysiological measurements show that the primary cultured cells have a low transepithelial resistance and high short-circuit current that was completely carried by [Na.sup.+] similar to a leaky epithelium like proximal tubule cells. This novel method established well-differentiated PTC cultures. aquaporin; electrophysiological characteristics; phloridzin; receptor-mediated endocytosis; sodium-dependent glucose transport; Ussing chamber
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- 2007
4. A highly reliable and budget-friendly Peltier-cooled camera for biological fluorescence imaging microscopy
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JOLLING, KOEN, VANDEVEN, MARTIN, VAN DEN EYNDEN, JIMMY, AMELOOT, MARCEL, and VAN KERKHOVE, EMMY
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- 2007
5. Maxi K+ channels in the basolateral membrane of the exocrine frog skin gland regulated by intracellular calcium and pH
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Andersen, Henning K., Urbach, Valerie, Van Kerkhove, Emmy, Prosser, Ena, and Harvey, Brian J.
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- 1995
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6. Electrophysiological evidence for the presence of an apical H+-ATPase in Malpighian tubules of Formica polyctena: intracellular and luminal pH measurements
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Zhang, Sheng -Lai, Leyssens, Anne, Van Kerkhove, Emmy, Weltens, Reinhilde, Van Driessche, Willy, and Steels, Paul
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- 1994
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7. A novel scorpion toxin blocking small conductance Ca 2+ activated K + channel
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Xu, Chen-Qi, He, Lin-Lin, Brône, Bert, Martin-Eauclaire, Marie-France, Van Kerkhove, Emmy, Zhou, Zhuan, and Chi, Cheng-Wu
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- 2004
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8. A novel scorpion toxin blocking small conductance Ca2+ activated K+ channel
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Xu, Chen-Qi, He, Lin-Lin, Brône, Bert, Martin-Eauclaire, Marie-France, Van Kerkhove, Emmy, Zhou, Zhuan, and Chi, Cheng-Wu
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- 2004
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9. PROXIMAL TUBULE CELLS IN PRIMARY CULTURE, A MODEL TO STUDY CHRONIC CADMIUM EXPOSURE AND INTERSTITIAL FIBROSIS. TRANSDIFFERENTIATION TO MYOFIBROBLASTS OCCURS IN THE PRESENCE OF TGF-β1
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Terryn, Sara, Vandenabeele, Frank, Moreels, Marjan, Steels, Paul, and Van Kerkhove, Emmy
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- 2003
10. The association between urinary kidney injury molecule 1 and urinary cadmium in elderly during long-term, low-dose cadmium exposure: a pilot study
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Dewitte Harrie, Reynders Carmen, Rigo Jean-Michel, Van Kerkhove Emmy, Nawrot Tim S, Munters Elke, De Winter Liesbeth M, Pennemans Valérie, Carleer Robert, Penders Joris, and Swennen Quirine
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kidney injury molecule 1 ,cadmium ,renal biomarkers ,toxicity ,chronic ,kidney ,Industrial medicine. Industrial hygiene ,RC963-969 ,Public aspects of medicine ,RA1-1270 - Abstract
Abstract Background Urinary kidney injury molecule 1 is a recently discovered early biomarker for renal damage that has been proven to be correlated to urinary cadmium in rats. However, so far the association between urinary cadmium and kidney injury molecule 1 in humans after long-term, low-dose cadmium exposure has not been studied. Methods We collected urine and blood samples from 153 non-smoking men and women aged 60+, living in an area with moderate cadmium pollution from a non-ferrous metal plant for a significant period. Urinary cadmium and urinary kidney injury molecule 1 as well as other renal biomarkers (alpha1-microglobulin, beta2-microglobulin, blood urea nitrogen, urinary proteins and microalbumin) were assessed. Results Both before (r = 0.20; p = 0.01) and after (partial r = 0.32; p < 0.0001) adjustment for creatinine, age, sex, past smoking, socio-economic status and body mass index, urinary kidney injury molecule 1 correlated with urinary cadmium concentrations. No significant association was found between the other studied renal biomarkers and urinary cadmium. Conclusions We showed that urinary kidney injury molecule 1 levels are positively correlated with urinary cadmium concentration in an elderly population after long-term, low-dose exposure to cadmium, while other classical markers do not show an association. Therefore, urinary kidney injury molecule 1 might be considered as a biomarker for early-stage metal-induced kidney injury by cadmium.
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- 2011
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11. Cadmium-Induced Pathologies: Where Is the Oxidative Balance Lost (or Not)?
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RAVINDRAN NAIR, Ambily, DEGHESELLE, Olivier, SMEETS, Karen, VAN KERKHOVE, Emmy, and CUYPERS, Ann
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Chemistry, Multidisciplinary ,cadmium ,oxidative stress ,antioxidants ,organ toxicity ,signalling ,cancer ,stem cells - Abstract
Over the years, anthropogenic factors have led to cadmium (Cd) accumulation in the environment causing various health problems in humans. Although Cd is not a Fenton-like metal, it induces oxidative stress in various animal models via indirect mechanisms. The degree of Cd-induced oxidative stress depends on the dose, duration and frequency of Cd exposure. Also the presence or absence of serum in experimental conditions, type of cells and their antioxidant capacity, as well as the speciation of Cd are important determinants. At the cellular level, the Cd-induced oxidative stress either leads to oxidative damage or activates signal transduction pathways to initiate defence responses. This balance is important on how different organ systems respond to Cd stress and ultimately define the pathological outcome. In this review, we highlight the Cd-induced oxidant/antioxidant status as well as the damage versus signalling scenario in relation to Cd toxicity. Emphasis is addressed to Cd-induced pathologies of major target organs, including a section on cell proliferation and carcinogenesis. Furthermore, attention is paid to Cd-induced oxidative stress in undifferentiated stem cells, which can provide information for future therapies in preventing Cd-induced pathologies. The authors apologize to any researcher whose work is not cited here due to limitations of space and scope. This work was supported by Hasselt University [BOF (Bijzonder onderzoeksfonds) projects; BOF08G01] through PhD grants for Ambily Ravindran Nair and Olivier DeGheselle. Additional funding came from tUL-impulsfinanciering (project toxicology), and Methusalem project (08M03VGRJ).
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- 2013
12. Cadmium-Induced Pathologies: Where Is the Oxidative Balance Lost (or Not)?
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Nair, Ambily, primary, DeGheselle, Olivier, additional, Smeets, Karen, additional, Van Kerkhove, Emmy, additional, and Cuypers, Ann, additional
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- 2013
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13. INCREASED MITOCHONDRIAL DNA-CONTENT ATTRIBUTABLE TO CADMIUM EXPOSURE AMONG ELDERLY
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Pieters, Nicky, primary, Munters, Elke, additional, Smeets, Karen, additional, Pennemans, Valerie, additional, Dewitte, Harry, additional, Penders, Joris, additional, Van Kerkhove, Emmy, additional, Vangronsveld, Jaco, additional, Cuypers, Ann, additional, and Nawrot, Tim, additional
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- 2011
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14. The association between urinary kidney injury molecule 1 and urinary cadmium in elderly during long-term, low-dose cadmium exposure: a pilot study
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Pennemans, Valérie, primary, De Winter, Liesbeth M, additional, Munters, Elke, additional, Nawrot, Tim S, additional, Van Kerkhove, Emmy, additional, Rigo, Jean-Michel, additional, Reynders, Carmen, additional, Dewitte, Harrie, additional, Carleer, Robert, additional, Penders, Joris, additional, and Swennen, Quirine, additional
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- 2011
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15. Cys-Loop Ligand-Gated Chloride Channels in Dorsal Unpaired Median Neurons ofLocusta migratoria
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Janssen, Daniel, primary, Derst, Christian, additional, Rigo, Jean-Michel, additional, and Van Kerkhove, Emmy, additional
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- 2010
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16. Low cadmium exposure triggers a biphasic oxidative stress response in mice kidneys
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Thijssen, Sandy, primary, Cuypers, Ann, additional, Maringwa, John, additional, Smeets, Karen, additional, Horemans, Nele, additional, Lambrichts, Ivo, additional, and Van Kerkhove, Emmy, additional
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- 2007
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17. Dorsal Unpaired Median Neurons ofLocusta migratoriaExpress Ivermectin- and Fipronil-Sensitive Glutamate-Gated Chloride Channels
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Janssen, Daniel, primary, Derst, Christian, additional, Buckinx, Roeland, additional, Van den Eynden, Jimmy, additional, Rigo, Jean-Michel, additional, and Van Kerkhove, Emmy, additional
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- 2007
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18. Exposing mice to low Cd doses triggers a biphasic oxidative stress response in the kidney: a role for Prdx2 and Nox4?
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Van Kerkhove, Emmy Valerie, primary, Cuypers, Ann, additional, Maringwa, John, additional, Horemans, Nele, additional, Lambrichts, Ivo, additional, and Thijssen, Sandy, additional
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- 2007
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19. Chronic exposure of mice to environmentally relevant, low doses of cadmium leads to early renal damage, not predicted by blood or urine cadmium levels
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Thijssen, Sandy, primary, Maringwa, John, additional, Faes, Christel, additional, Lambrichts, Ivo, additional, and Van Kerkhove, Emmy, additional
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- 2007
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20. Midgut electrophysiology of a small insect Orchesella cincta (L.): regional differences and influence of molting
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Van Kerkhove, Emmy, primary and Klein, Georg, additional
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- 2006
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21. Problems and pitfalls in cable analysis in short tubular epithelia. Analysis of regional functional differences, a model
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Klein, Georg, primary and Van Kerkhove, Emmy, additional
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- 2006
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22. BmBKTx1, a Novel Ca2+-activated K+ Channel Blocker Purified from the Asian Scorpion Buthus martensi Karsch
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Xu, Chen-Qi, primary, Brône, Bert, additional, Wicher, Dieter, additional, Bozkurt, Özlem, additional, Lu, Wu-Yuan, additional, Huys, Isabelle, additional, Han, Yu-Hong, additional, Tytgat, Jan, additional, Van Kerkhove, Emmy, additional, and Chi, Cheng-Wu, additional
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- 2004
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23. Body extracts from the forest ant and honeybee: chromatographic comparison of factors affecting the ant Malpighian tubule
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Laenen, Bart, primary, Wiehart, Ursula, additional, De Decker, Nadine, additional, Steels, Paul, additional, and Van Kerkhove, Emmy, additional
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- 2003
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24. Effect of Bacillus thuringiensis Cry1 Toxins in Insect Hemolymph and Their Neurotoxicity in Brain Cells of Lymantria dispar
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Cerstiaens, Anja, primary, Verleyen, Peter, additional, Van Rie, Jeroen, additional, Van Kerkhove, Emmy, additional, Schwartz, Jean-Louis, additional, Laprade, Raynald, additional, De Loof, Arnold, additional, and Schoofs, Liliane, additional
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- 2001
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25. An antidiuretic factor in the forest ant: purification and physiological effects on the Malpighian tubules
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Laenen, Bart, primary, De Decker, Nadine, additional, Steels, Paul, additional, Van Kerkhove, Emmy, additional, and Nicolson, Sue, additional
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- 2001
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26. Partial identification of a peptide that stimulates the primary urine production of single isolated Malpighian tubules of the forest ant, Formica polyctena
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Laenen, Bart, primary, Verhaert, Peter, additional, Schoofs, Liliane, additional, Steels, Paul, additional, and Van Kerkhove, Emmy, additional
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- 1999
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27. Eicosanoid biosynthesis inhibitors modulate basal fluid secretion rates in the Malpighian tubules of the ant, Formica polyctena
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Van Kerkhove, Emmy, primary, Pirotte, Patrick, additional, Petzel, David H., additional, and Stanley-Samuelson, David W., additional
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- 1995
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28. Inward-rectifier potassium channels in basolateral membranes of frog skin epithelium.
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Urbach, Valerie and Van Kerkhove, Emmy
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POTASSIUM channels , *ELECTRIC properties of biological membranes , *EPITHELIUM , *PHYSIOLOGY - Abstract
Examines the inward-rectifier potassium channels in basolateral membranes (BLM) of frog skin epithelium. Identification of potassium channel responsible for potassium recycling across BLM; Current-voltage relationship of BLM; Causes of inward rectification; Generation of BLM conductance by inward-rectifier potassium channel under physiological ionic gradients.
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- 1994
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29. Changes in expression of fibrotic markers and histopathological alterations in kidneys of mice chronically exposed to low and high Cd doses
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Thijssen, Sandy, Lambrichts, Ivo, Maringwa, John, and Van Kerkhove, Emmy
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CADMIUM , *KIDNEYS , *FIBROSIS , *LABORATORY mice - Abstract
Abstract: The main target organ for cadmium (Cd) is the kidney, and more specifically the proximal tubular cells. Little is known about the effects of a long-term Cd exposure on the ultrastructure of the kidney and the involvement in tubulointerstitial fibrosis. Therefore, mice were exposed to Cd concentrations varying from 10 to 500mg CdCl2/l in the drinking water during 4, 16 and 23 weeks. Ultrastructural changes were studied by means of light- and electron microscopical analyses. Furthermore, the expression of the extracellular matrix (ECM) proteins collagen I and fibronectin, and the myofibroblast/epithelial-to-mesenchymal transition (EMT) marker alfa-smooth muscle actin (α-SMA) were studied by means of immunohistochemistry. The histopathological changes caused by Cd varied considerably from one animal to another, and from one individual cell to another. An exposure to Cd concentrations up to 100mg CdCl2/l elicited only minor changes that were restricted to increasing amounts of lysosomes and vacuolisation. When higher Cd concentrations were applied, the changes became more pronounced and featured mitochondrial damage, cellular swelling and loss of basal invaginations. An overproduction of the interstitial matrix component fibronectin and the expression of the myofibroblasts/EMT marker α-SMA in kidneys of mice exposed to 100mg CdCl2/l clearly indicated that an exposure to relatively low Cd doses might lead ultimately to renal fibrosis. Increasing the Cd dose (up to 500mg CdCl2/l) evoked an increased immunoreactivity for fibrotic markers. In conclusion we may state that concentrations up to 100mg CdCl2/l evoked minor changes, although the expression of fibrotic markers was increased. Changes became more pronounced when exposing to higher Cd concentrations. [Copyright &y& Elsevier]
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- 2007
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30. BmBKTx1, a Novel Ca2+-activated K+ Channel Blocker Purified from the Asian Scorpion Buthus martensi Karsch.
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Chen-Qi Xu, Brône, Bert, Wicher, Dieter, Bozkurt, Özlem, Wu-Yuan Lu, Huys, Isabelle, Yu-Hong Han, Tytgat, Jan, Van Kerkhove, Emmy, and Cheng-Wu Chi
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TOXINS , *CALCIUM antagonists , *ION channels , *BUTHUS , *NEURONS , *BIOCHEMISTRY , *MIGRATORY locust , *DROSOPHILA - Abstract
BmBKTx1 is a novel short chain toxin purified from the venom of the Asian scorpion Buthus martensi Karsch. It is composed of 31 residues and is structurally related to SK toxins. However, when tested on the cloned rat SK2 channel, it only partially inhibited rSK2 currents, even at a concentration of 1 μM. To screen for other possible targets, BmBKTx1 was then tested on isolated metathoracic dorsal unpaired median neurons of Locusta migratoria, in which a wide variety of ion channels are expressed. The results suggested that BmBKTx1 could specifically block voltage-gated Ca2+-activated K + currents (BK-type). This was confirmed by testing the BmBKTx1 effect on the α subunits of BK channels of the cockroach (pSlo), fruit fly (dSlo), and human (hSlo), heterologously expressed in HEK293 cells. The IC50 for channel blocking by BmBKTx1 was 82 nM for pSlo and 194 nM for dSlo. Interestingly, BmBKTx1 hardly affected hSlo currents, even at concentrations as high as 10 μM, suggesting that the toxin might be insect specific. In contrast to most other scorpion BK blockers that also act on the Kv1.3 channel, BmBKTx1 did not affect this channel as well as other Kv channels. These results show that BmBKTx1 is a novel kind of blocker of BK-type Ca2+-activated K+ channels. As the first reported toxin active on the Drosophila Slo cbAnnel dSlo, it will also greatly facilitate studying the physiological role of BK channels in this model organism. [ABSTRACT FROM AUTHOR]
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- 2004
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31. A novel scorpion toxin blocking small conductance Ca2+ activated K+ channel
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Xu, Chen-Qi, He, Lin-Lin, Brône, Bert, Martin-Eauclaire, Marie-France, Van Kerkhove, Emmy, Zhou, Zhuan, and Chi, Cheng-Wu
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SCORPION venom , *TOXINS , *AMINO acids , *PEPTIDES - Abstract
Small conductance calcium activated potassium channels (SK) are crucial in the regulation of cell firing frequency in the nervous system and other tissues. In the present work, a novel SK channel blocker, designated BmSKTx1, was purified from the scorpion Buthus martensi Karsh venom. The sequence of the N-terminal 22 amino acid residues was determined by Edman degradation. Using this sequence information, the full-length cDNA and genomic gene of BmSKTx1 were cloned and sequenced. By these analyses, BmSKTx1 was found to be a peptide composed of 31 amino acid residues with three disulfide bonds. It shared little sequence homology with other known scorpion α-KTxs but showed close relationship with SK channel blockers in the phylogenetic tree. According to the previous nomenclature, BmSKTx1 was classified as α-KTx14.1. We examined the effects of BmSKTx1 on different ion channels of rat adrenal chromaffin cells (RACC) and locust dorsal unpaired median (DUM) neurons. BmSKTx1 selectively inhibited apamin-sensitive SK currents in RACC with
Kd of 0.72 μM and Hill coefficient of 2.2. And it had no effect on Na+, Ca2+, Kv, and BK currents in DUM neuron, indicating that BmSKTx1 was a selective SK toxin. [Copyright &y& Elsevier]- Published
- 2004
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32. Validation and evaluation of novel biomarkers for early renal damage in the clinical setting
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Pennemans, Valerie, Penders, Joris, Swennen, Quirine, and Van Kerkhove, Emmy
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The aim of this thesis was to contribute to the next step in the implementation of these new biomarkers for AKI. In chapter 2 and chapter 3, we focused on the determination of the most ideal collection and storage conditions prior to the biomarker measurements. In chapter 4 the goal was to establish reference values for these novel biomarkers for healthy subjects with special attention for age and gender. In the following chapters we investigated the utility of these biomarkers in different clinical settings. Chapter 5 was attributed to the research we conducted on the association between KIM-1 and urinary cadmium concentrations. Finally, in chapter 6 we investigated a panel of novel biomarkers in an attempt to detect AKI after cardiac surgery requiring a cardiopulmonary bypass. Momenteel is het vroegtijdig opsporen van acuut en chronisch nierfalen gebaseerd op de stijging van de creatinine concentratie in het serum in combinatie met een daling in urine volume. Deze detectiemethode heeft het nadeel dat de vermelde veranderingen pas enkele uren tot dagen na het eigenlijk ontstaan van het nierprobleem waargenomen kunnen worden. Er is dan ook nood aan nieuwe, snellere detectiemethoden of biomerkers. Via proteomische onderzoeksmethoden werden een aantal eiwitten in het bloed en de urine ontdekt, die mogelijkerwijze als biomerker voor nierschade kunnen dienen. Deze eiwitten blijken in experimenten bij dieren en mensen immers verhoogd tot expressie te komen in geval van nierschade. Deze nieuwe biomerkers dienen echter gevalideerd te worden: er moet nagegaan worden op welke wijze ze het best gemeten worden, wat normale waarden zijn bij gezonde mensen en bij welke pathologieën en in welke patiëntengroepen ze verhoogd zijn. In deze doctoraatsthesis hebben we ons gericht op het validatieproces van enkele nieuwe biomerkers voor nierschade, nl. KIM-1, NGAL, NAG en Cystatine C. We hebben vooreerst aangetoond dat KIM-1, gemeten in de urine, niet afhankelijk is van de pH van de urine, maar de commerciële kit die gebruikt wordt voor de meting wel. Het is dan ook van belang om urinestalen voor de meting van KIM-1 op een pH rond 7 te brengen (hoofdstuk 2). Enkele andere verzamel- en opslagspecificaties voor de urinestalen waarin de biomerkers gemeten worden (zoals het toevoegen van protease inhibitoren, centrifugatie, maximale duur voor invriezen en de invriestemperatuur en –periode) zijn vervolgens onder de loep genomen, en richtlijnen hieromtrent zijn uitgewerkt (hoofdstuk 3). In het volgende deel van ons onderzoeksproject, hebben we urinestalen van verondersteld gezonde proefpersonen van alle leeftijden verzameld om zo referentiewaarden voor KIM-1, NGAL, Cystatine C en NAG op te stellen (hoofdstuk 4). Uit dierproeven van andere onderzoeksgroepen werd een positieve associatie afgeleid tussen de blootstelling aan het zware metaal cadmium, waarvan geweten is dat het schadelijke effecten heeft op de nier enerzijds en verhoogde KIM-1 concentraties in de urine anderzijds. We onderzochten een proefgroep van oudere personen die wonen in de omgeving van een industrieterrein met gekende verhoogde cadmiumwaarden in de bodem en de lucht en dus gedurende een lange periode blootgesteld zijn aan cadmium. Eenzelfde associatie werd aangetoond tussen cadmium en KIM-1 in de urine van deze proefpersonen (hoofdstuk 5). In het laatste deel van dit doctoraat werd onderzocht of de nieuwe biomerkers een rol kunnen spelen in de vroege detectie van acuut nierfalen, na het ondergaan van cardiale chirurgie waarbij extracorporele circulatie noodzakelijk is. Hierbij werden de concentraties van de urinaire biomerkers voor en op verschillende tijdstippen na de operatie vergeleken tussen volwassen patiënten die acuut nierfalen hadden ontwikkeld of niet. Voor elke gemeten biomerker (urinair KIM-1, NGAL, cystatine C en serum cystatine C) kon een associatie met acuut nierfalen gevonden worden op tenminste één tijdstip. Alleen serum Cystatine C bleek echter een voorspellende meerwaarde te hebben bovenop een klinisch model, dat werd samengesteld uit klinische parameters die momenteel reeds gebruikt worden in de detectie van AKI in het ziekenhuis (hoofdstuk 6).
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- 2014
33. Cadmium-induced nephrotoxicity: from defense strategy to acclimation
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Ravindran Nair, Ambily, Cuypers, Ann, Smeets, Karen, and Van Kerkhove, Emmy
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Cadmium (Cd) indirectly induces reactive oxygen species (ROS) by (1) a displacement of redox-active metals, (2) depletion of redox scavengers, (3) inhibition of anti-oxidant enzymes and (4) inhibition of the electron transport chain. This ultimately results in mitochondrial damage leading to loss of function or cell death in multiple organs. A disturbance of the redox balance by Cd at the cellular level has been studied repeatedly in different experimental set-ups including differentiated cells at the whole animal level (including humans), tissue level, primary cell cultures and/or cell lines as well as non-differentiated cells. However the outcome of these results as to where the oxidative balance gets compromised at the cellular level and leads to pathologies at the level of the organ depends on a multitude of experimental and environmental conditions. It is important to keep in mind that the comparison of results from different experiments should be done with caution as many studies use high concentrations of Cd that might cause an effect in cell lines, but are irrelevant in terms of environmentally realistic exposures in animals. Several parameters that are inherently connected to the experimental set-up such as the route of Cd administration, the speciation of Cd applied and different cell types to be studied all determine the final outcome of Cd toxicity. In my study, research on kidney cell lines for example, results in direct exposure of Cd on these cells, while an oral or sub-cutaneous administration of Cd to rats in vivo results in Cd absorption by blood and other organs before it reaches the kidney resulting in changes of initial Cd speciation and concentration applied. Nevertheless, in terms of translational toxicology, it is highly interesting to compare the effects of Cd exposure levels in vitro to those in vivo, when Cd concentrations are determined for both experimental set-ups in a comparable manner. Cadmium has a half-life of 15-30 years in the kidney, and considerable damage is mostly confined to the apical domain of the proximal tubular cells (PTCs). These cells possess large amounts of mitochondria, an important source of ROS, but also a highly sensitive organelle to increased ROS levels. As such, mitochondrial alterations are involved in both damaging as well as adaptation responses. This makes the WKPT-0293 cell line derived from the S1 segment of PTCs of the rat a good model to study Cd toxicity, as they also possess transporters and receptors for Cd. The mechanism of Cd-induced ROS production in association with mitochondrial alterations leading to adaptation responses or cell death is not fully elucidated, allthough it is known that Cd can directly damage mitochondria. In general, the aim of this study was to investigate and compare the molecular and cellular responses in renal cells in vitro and in vivo exposed to different doses of Cd in an acute respectively subchronic way. The study specifically focused on the role of oxidative stress and mitochondria, and how they modulate different defense strategies and help cells to acclimate to Cd stress. Gene expression analyses of antioxidant genes and mitochondrial genes formed the core technology of this study and therefore it was very important that the results obtained from qPCR are accurate and reliable. For this purpose, an additional analysis to select appropriate reference genes was performed in both cell lines and kidney tissues of in vitro and in vivo experiments respectively. The choice of reference genes for gene quantification is an important pre-requisite for carrying out new studies. From a set of eight commonly used reference genes, it was found that glyceraldehyde-3-phosphate dehydrogenase (Gapdh), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (Ywhaz) and beta-actin (Actb) were the most stable reference genes in the in vitro experimental set-up, while Gapdh, Ywhaz and peptidylprolyl isomerase A /cyclophilin A (Ppia) were the stable set in vivo. It was shown that the stability and hence selection of reference genes varied between both experimental set-ups even when both the cells/tissues originated from rat. Subsequently all the qPCR results of transcripts analyzed in vitro or in vivo were normalized to their respective set of stable reference genes. The in vitro experiment made use of Cd free medium and of 1, 10 and 30 μM of Cd concentrations that caused low, moderate or high amounts of damage to PTCs. In this case, cells without or with visible alterations were obtained to study the antioxidant signature and role of mitochondria during Cd exposure and/or subsequent adaptation. It was shown that at lower Cd concentrations (1 μM CdCl2), mitochondrial DNA (mtDNA) content and anti-apoptotic gene expression increased with a decrease in reduced glutathione (GSH) content. At higher concentrations (10 and 30 μM CdCl2), there was an increase in hydrogen peroxide (H2O2) and glutathione disulfide (GSSG) content, metallothionein (MT) transcript levels, pro-apoptotic gene induction together with loss of mitochondrial DNA content and function as well as depletion of GSH. From these observations, we noticed that alterations in the balance between the pro- and anti-oxidant status of the cell can lead to cell signalling for survival or damage to cells, called the oxidative challenge. With increasing ROS and thereby cell stress, the mitochondrial system can collapse, leading to further damage of mitochondria and ROS production culminating in apoptosis and/or necrosis. Both mitochondrial abundance and a shift from GSH to GSSG as a response to Cd induced oxidative stress are probably involved in the final outcomes towards long-term exposure and were explored in more detail in the in vivo study. In the in vivo study, when rats were treated for 13 days with sub-chronic Cd exposure (1 mg CdCl2/kg bw), the GSSG/GSH ratio remained the same indicating the maintenance of the cellular redox state. Superoxide dismutase 2 and GSH immunohistochemical stainings together with gene expression analyses of antioxidants revealed their up-regulated activities, which points towards the cell’s attempt to defend, and adapt to the Cd-induced oxidative challenge. It is clear that under Cd stress, a new redox equilibrium is installed in the PTCs. The increased expression of genes responsible for regulation and biogenesis of mitochondria indicates a positive regulation for mitochondrial biogenesis. This was also reflected in terms of increased mtDNA content in Cd-treated animals as compared to control animals, which might be the result of an increasing energy demand of the cell necessary for adaptive and or survival mechanisms under Cd stress. Whereas increased caspase activity in Cd-treated animals points towards enhanced apoptosis under Cd stress, the simultaneous up-regulation of both pro- and antiapoptotic genes might represent the varying activities of damaged and undamaged cells present in the heterogeneous kidney tissue. Overall, the in vivo experimental results suggested a clear and obvious involvement of mitochondria in defending against the Cd-induced oxidative stress during long-term subchronic exposure. In conclusion, a comparison of the oxidative stress signature between acute in vitro versus sub-chronic in vivo Cd exposure shows a clear shift in terms of defense and acclimation. It was shown that GSH is a first line of defense in Cd toxicity both in vitro and in vivo due to its chelating and antioxidant properties. There is also a parallel activation of mitochondrial biogenesis in order to meet the increasing energy demand of the cells under stress. Other acclimation strategies i.e. chelation by MTs and antioxidant defense mechanisms come into play during exposure to higher Cd concentrations or prolonged exposure. Once the Cd stress is beyond the withholding limit, these mechanisms fail and the cell enters a point of no return leading to irreversible damage and eventually Cdinduced pathologies. Cadmium (Cd) veroorzaakt het ontstaan van ROS (reactive oxygen species) op een indirecte manier door (1) het verdringen van redox-actieve metalen, (2) de depletie van antioxidatieve metabolieten, (3) de inhibitie van antioxidatieve enzymen en (4) de inhibitie van de elektronentransportketen. Dit resulteert uiteindelijk in schade aan de mitochondriën, wat leidt tot functieverlies, en uiteindelijk celdood in verschillende organen. Een verstoring van de redoxbalans door Cd op cellulair niveau werd al herhaalde malen bestudeerd in verschillende experimentele set-ups zoals gedifferentieerde cellen in dier (en mens), op weefselniveau, in primaire celculturen en/of in cellijnen en ook in nietgedifferentieerde cellen. Nochtans, of en waar de oxidatieve balans al dan niet in het gedrang komt en leidt tot pathologieën t.h.v. de organen hangt af van allerlei experimentele alsook omgevingsfactoren. Het is belangrijk de vergelijking van resultaten uit verschillende experimenten met de nodige omzichtigheid uit te voeren en rekening te houden met het feit dat vele studies hoge concentraties van Cd gebruiken, die wel een effect kunnen hebben op cellijnen, maar irrelevant zijn in termen van de reële blootstelling aan Cd van mens en dier in het milieu. Verschillende parameters in een bepaalde experimentele set-up, zoals de blootstellingsroute, de gebruikte Cd speciatie, het soort celtype zijn allen van invloed op de uiteindelijke uitkomst en op de gemeten Cd toxiciteit. In mijn studie, in het onderzoek op renale cellijnen bij voorbeeld werden cellen direct blootgesteld aan Cd, terwijl - na oraal of subcutaan toedienen van Cd aan ratten in vivo - Cd eerst in het bloed en in andere organen wordt opgenomen, vooraleer het de nier bereikt. Dit veroorzaakt een wijziging in de speciatie van Cd en van de concentratie, waaraan de niercellen uiteindelijk worden blootgesteld. Nochtans is het - in termen van translationele toxicologie – erg interessant om de effecten van het niveau van Cd blootstelling in vitro en in vivo te vergelijken, wanneer de Cd concentraties in beide experimentele set-ups vergelijkbaar zijn. Cadmium heeft een halfleven van 15-30 jaar in de nier. Aanzienlijke schade is meestal beperkt tot het apicale gedeelte van de proximale tubuluscellen (PTCs). Deze cellen beschikken over een groot aantal mitochondriën, een belangrijke bron van ROS, maar zelf ook erg gevoelig aan een verhoogde aanwezigheid van ROS. Veranderingen in de mitochondriën zijn betrokken bij zowel het veroorzaken van schade als in aanpassingsreacties. De WKPT-0293 cellijn, afgeleid van het S1 segment van PCTs van de rat, is een goed model voor de studie van Cd toxiciteit, want de cellen beschikken over transporters en receptoren voor Cd. Het mechanisme van Cd-geïnduceerde ROS productie en de samenhang met mitochondriale veranderingen, die tot adaptatie leiden, werden nog niet helemaal opgehelderd, al is wel al geweten dat Cd de mitochondriën rechtstreeks kan beschadigen. Kort samengevat, was het doel van deze studie om de moleculaire en cellulaire responsen te bestuderen en te vergelijken van niercellen in vitro en in vivo en dit bij blootstelling aan verschillende dosissen van Cd op een acute en een sub-chronische manier. De focus van de studie lag op de rol van oxidatieve stress en van de mitochondriën en hoe deze verschillende verdedigingsstrategiën moduleren om cellen te helpen om zich aan te passen aan Cd stress. De belangrijkste techniek, gebruikt in deze studie, betrof analyses van genexpressie van antioxidante en mitochondriale genen. Het was dus erg belangrijk om accurate en betrouwbare qPCR resultaten te bekomen. Om die reden werd een voorafgaande analyse gedaan van nier cellijnen en van nierweefsel in vitro en in vivo, dit om de best geschikte referentiegenen op te sporen. Een juiste keuze van referentiegenen om andere genen te kunnen kwantificeren is erg belangrijk, wanneer men een nieuwe studie aanvat. Van de acht genen die meestal gebruikt worden als referentiegenen waren het vooral glyceraldehyde-3-fosfaat dehydrogenase (Gapdh), tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activatie proteine, zeta polypeptide (Ywhaz) en beta-actine (Actb) die het meest stabiel waren in de in vitro set-up, terwijl Gapdh, Ywhaz en peptidylprolyl isomerase A /cyclophilin A (Ppia) stabiel waren in de in vivo experimenten. We konden aantonen dat de stabiliteit en dus de selectie van de referentiegenen verschilde tussen beide experimentele set-ups, ook al was de rat telkens de bron van de cellen of weefsels. Alle qPCR resultaten van gentranscripten, die in vitro en in vivo werden geanalyseerd, werden vervolgens genormaliseerd naar hun respectievelijke set van stabiele referentiegenen. In de in vitro experimenten werden, naast Cd-vrij medium ter controle, concentraties van 1, 10 en 30 μM Cd toegepast. Deze concentraties brachten respectievelijk lage, middelmatige en hoge schade toe aan de PTCs. Op die manier werden cellen bekomen zonder en met zichtbare veranderingen, die we konden gebruiken om de antioxidatieve respons en de rol van de mitochondriën te bestuderen gedurende de Cd blootstelling en/of de daarop volgende adaptatie. Bij lagere Cd concentraties (1 μM CdCl2) steeg de hoeveelheid mitochondriaal DNA (mtDNA) en de expressie van anti-apoptotische genen verhoogde, terwijl het niveau van gereduceerd glutathion (GSH) verlaagd was. Bij hogere concentraties (10 en 30 μM CdCl2) was er een stijging van de hoeveelheid waterstof peroxide (H2O2) en van glutathiondisulfide (GSSG), van het metallothioneïne (MT) transcriptie niveau en was er een verhoogde inductie van pro-apoptotische genen, tezamen met een verlies aan mitochondriale DNA inhoud en functie, evenals een depletie van GSH. Uit deze resultaten werd duidelijk dat veranderingen in de pro- en anti-oxidant status van de cel kunnen leiden tot celsignalisatie, met als gevolg ofwel het overleven van de cel, ofwel celschade, de zogenaamde ‘oxidative challenge’. Bij verhoogde ROS en dus cel stress kan het mitochondriaal systeem falen, waardoor verdere schade van de mitochondriën ontstaat en verdere ROS productie, resulterend in apoptose en/of necrose van cellen. Zowel de hoeveelheid mitochondriën, als een verschuiving van GSH naar GSSG als respons op de Cd-geïnduceerde oxidatieve stress zijn waarschijnlijk betrokken bij het uiteindelijk resultaat bij lange termijn blootstelling en werden meer in detail bestudeerd in de in vivo studie. In de in vivo studie werden ratten gedurende 13 dagen sub-chronisch blootgesteld aan 1mg Cd/kg lichaamsgewicht. De GSSG/GSH ratio bleef constant, wat betekent dat de cellulaire redox status werd gehandhaafd. Superoxide dismutase 2 en GSH immunohistochemische kleuringen, gecombineerd met analyse van genexpressie van antioxidanten toonden aan dat de activiteit verhoogd was, wat wijst op een poging van de cel om zich te verdedigen en zich aan te passen aan de Cd-geïnduceerde toxiciteit. Het is duidelijk dat er bij Cd stress een nieuw redox evenwicht ontstond in de PTCs. De verhoogde expressie van genen, verantwoordelijk voor de regeling en biogenese van mitochondriën een gunstige bijsturing van de biogenese van mitochondrën. De verhoogde mtDNA inhoud in dieren behandeld met Cd, vergeleken met controledieren, wees in dezelfde richting. Mogelijkerwijze wijst dit op een verhoogde behoefte aan energie, nodig voor de aanpassings- en/of de overlevingsmechanismen van de aan Cd-blootgestelde cellen. Terwijl de verhoogde caspase activiteit in de aan Cd-blootgestelde dieren wijst op een verhoogde apoptose, kan de gelijktijdige op-regulatie van zowel pro- als anti-apoptotische genen te wijten zijn aan de heterogeniteit van het nierweefsel en bijgevolg de aanwezigheid van zowel beschadigde als niet beschadigde cellen. In elk geval wijzen de in vivo resultaten op een duidelijke rol van de mitochondriën in de verdediging tegen Cd-geïnduceerde oxidatieve stress bij sub-chronische, langdurige blootstelling. Tenslotte kunnen we besluiten dat de vergelijking van de oxidatieve stress respons in in vitro versus sub-chronische in vivo Cd blootstelling wijst op een duidelijke verschuiving voor wat betreft verdediging en aanpassing. Er werd aangetoond dat GSH, door zijn bindende en zijn antioxidatieve eigenschappen, de eerste lijnsdefensie vertegenwoordigt zowel in vitro als in vivo. Er was ook een parallelle activatie van mitochondriale biogenese om aan de verhoogde energiebehoefte van cellen onder stress te kunnen voldoen. Andere aanpassingsstrategieën zoals binding door MTs en antioxidatieve verdedigingsmechanismen beginnen een rol te spelen bij blootstelling aan hogere Cd concentraties of bij langere blootstelling. Eens de Cd stress voorbij de grens van deze mechanismen gaat, bereiken de cellen een ‘point of no return’, wat eindigt in irreversibele schade en uiteindelijk in Cd-geïnduceerde pathologiën.
- Published
- 2013
34. Cys-loop ligand-gated chloride channels in Dorsal Unpaired Median neurons of Locusta migratoria
- Author
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Janssen, Daniel, Van Kerkhove, Emmy, and Rigo, Jean-Michel
- Published
- 2009
35. A primary culture of proximal tubule cells of the mouse kidney on collagen coated membranes: characterization, electrophysiological properties and response to stress factors
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TERRYN, Sara, Van Kerkhove, Emmy, and Steels, Paul
- Abstract
Over the years, a large number of in vitro models ranging from renal cell lines to isolated perfused tubules have been established. However, epithelial cell lines had to undergo a number of genetic changes in order to survive indefinitely in vitro. On the other hand, the isolated perfused tubule is a complex system to study. As a consequence, primary epithelial cell culture systems are needed to check the physiological significance of studies done with established cell lines and in some cases they might be the only way to study specific tubular cell function characteristics in particular (patho)physiological and pharmacological conditions. Furthermore, the increasing use of transgenic and knock-out mouse models should be supported by an in vitro model to study the cellular phenotype. Major inherent technical advantages of cultured cells for the study of epithelial transport include their ability to be studied in a controlled environment without systemic neurons and hormonal influences, and their easy accessibility for electrophysiological and imaging techniques. However, primary cell culture also has its limitations such as incomplete differentiation of cells or a limited lifespan, among others. The objective of this thesis was to establish a primary cell culture of mouse proximal tubule cells. The primary cells were extensively characterized for their proximal tubule origin. To correlate our findings in primary cell cultures with data in the literature, a detailed description of proximal tubule cell morphology and ( electro-) physiology is given in section l. 2, l. 3 and 1.4. Finally, we used our cell cultures as a model for the study of renal tubulointerstitial fibrosis. The mechanisms involved in tubulointerstitial fibrosis are described in section 1.5. At the end of this chapter, the different aims of this thesis are summarized.
- Published
- 2007
36. Midgut electrophysiology of Orchesella cincta (L.) Templeton 1835
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Klein, Georg and Van Kerkhove, Emmy
- Abstract
The objectives of this work were: To study some functional properties of the midgut. 1) First of all some essential information was needed on the electrophysiologica l properties of the midgut of 0. cincta. la) To start with, an elemental analysis of 0. cincta hemolymph was performed and an adequate physiological saline was composed, based on the hemolymph composition. lb) Furthermore we wanted to study the electrical potential differences at the basolateral and the apical membrane, i.e. to assess the importance of the conductance of the basolateral membrane for K+ and to make an estimate of the relative basolateral versus apical membrane resistance. If the basolateral resistance is lower than the apical resistance Vbi approximates Ebi (see chapter 1.2.4 ). If the basolateral resistance can be increased, for instance by the application of Ba2+, we could take advantage of this situation to learn more about the electrical events at the apical membrane. (electrogenic pumping mechanisms or conductances of other ions). lc) The measurement of pH in the hemolymph, in the cell and in the lumen, should allow to calculate the electrochemical gradient across the basolateral and the apical membranes. This is important information because pH is an important factor either regulating transporters or offering a gradient across the cell membrane, energizing secondary active transport. The items la, lb and le are covered in chapter IA. 2) to determine the specific transepithelial resistance (Rte) of the midgut epithelium. Despite its small size, the midgut of Orchesella cincta must fulfill all needs of digestion and uptake of nutrients. Inevitably, the composition of the food composition changes as a consequence of digestion and uptake of food, while it moves through the midgut. Transport processes in the epithelium might therefore be adapted to a gradual change in the luminal content at different sites along the midgut. Consequently the Rte might be unequally distributed along the midgut. Because of t he small size of the animal t he Rte had to be determined with cable analysis. One assumption of cable analysis is, that the Rte is uniformly distributed along the tissue. 2a) Because this may not necessarily be the case, we tried to evaluate, whether it is nevertheless possible to find a good estimate of the true Rte if it changes along the midgut and 2b) whether it is possible to distinguish between artifacts due to electrical leakage in the set-up and a true unequal distribution of R,, along the midgut. The items outlined in 2), 2a) and 2b) are covered in chapter IB. 3) Furthermore, a technique for direct measurement of Cd2+ fluxes was introduced at the lab to explore the possibility of a simple self-referencing ion-selective electrode set-up to detect Cd2+ fluxes at the basolateral side of the midgut, which is known to accumulate cadmium.
- Published
- 2003
37. DUM neurons of the locust: electrophysiological properties and effect of invertebrate toxins
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Brône, Bert and Van Kerkhove, Emmy
- Published
- 2003
38. Purification, characterization and mode of action of endogenous neuroendocrine factors in the forest ant, formica polyctena
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LAENEN, BART, Van Kerkhove, Emmy, and Steels, Paul
- Abstract
The Malpighian tubules of the forest ant, F. polyctena, have been studied for many years now by the research group physiology of the Limburgs Universitair Centrum (Diepenbeek, Belgium). In insects these tubules are responsible for the primary urine production. The latter is achieved via secretion, driven by the active transport of KCI from hemolymph to lumen, and passive diffusion of substances. Initially the transport mechanisms involved in this active transport have been investigated. In a subsequent phase the factors involved in the regulation of this active transport were investigated, i.e. the presence of endogenous factors with a direct effect on the primary urine production by the Malpighian tubules was investigated. Both diuretic and antidiuretic factors were found to be present in body extracts of the ants. Some of these factors, i.e. a diuretic peptide (Fop-DP I) and two myotropic peptides (Fop-TK I en Fop-TK II), could be purified. In the present study we tried to further complete this picture. Different factors, both diuretic (Fop-DP and Fop-GP) and antidiuretic (FopADF), were shown to be present in body extracts of the ant and some could be identified. The antidiuretic factor, purified from an abdomen extract, was shown to block the Transport mechanisms involved in the active transport of K+ through the cells of the Malpighian tubules (see chapter 1). The signal transduction pathway used by FopADF could not be clarified. The involvement of adenylate cyclase and / or an increase in intracellular cAMP could clearly be excluded (see chapter 2). A cross-reactivity of Tenebrio molitor Malpighian tubules towards FopADF could be demonstrated by Dr. S. Nicolson (see Note after chapter 1) and a similar antidiuretic factor was shown to be present in an abdomen extract of the honeybee, Apis mellifer (see chapter 4). The diuretic factors, both purified from head/ thorax extracts, were shown to be peptides. For Fop-DP approximately 35% of the amino acid sequence could be determined. Fop-DP acted via an increase in intracellular calcium (see Chapter 3). A Fop-DP related factor was also demonstrated to be present in a head extract of the honeybee (see chapter 4). The second diuretic factor, Fop-cGP, was fully characterized. It is a peptide of 30 amino acids which exerts its diuretic effect in Locust, via an increase in cAMP (see Chapter 6). The purification of Fop-cGP followed a preliminary study trying to establish the presence of CRF-related peptides in the forest ant. The latter were shown to be present in a head / thorax extract of the ant by means of chromatography and an ELISA based on polyclonal antisera to CRF-related peptides from other insects (see Chapter 5). Fop-cGP had the activity of a CRF-like factor but not the amino acid composition. Another factor having a CRF-like structure may be present but still awaits further purification and characterization. Below we tried to give a brief overview of the relevant results for each chapter. A table, added as bookmarker, summarizes the results and is intended as a reader guide. The factors involved in the endocrine regulation of the primary urine production of the forest ant is compared to the situation known for other insects.
- Published
- 1998
39. Enkele moraalfilosofische beschouwingen bij De belofte aan Rachel
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Johan Stuy, Centre for Ethics, Vrije Universiteit Brussel, Bastiaensen, Guy, Lampo, Jan, Van Kerkhove, Emmy, History, Archeology, Arts, Philosophy and Ethics, and Centrum voor Ethiek en Humanisme
40. Cys-loop ligand-gated chloride channels in dorsal unpaired median neurons of Locusta migratoria.
- Author
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Janssen D, Derst C, Rigo JM, and Van Kerkhove E
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Chloride Channels genetics, Glutamic Acid metabolism, Histamine metabolism, In Vitro Techniques, Insect Proteins genetics, Locusta migratoria, Membrane Potentials drug effects, Molecular Sequence Data, Neurons cytology, Neurons drug effects, Patch-Clamp Techniques, Reverse Transcriptase Polymerase Chain Reaction, Sequence Analysis, DNA, Sequence Homology, Time Factors, gamma-Aminobutyric Acid metabolism, Chloride Channels metabolism, Insect Proteins metabolism, Neurons physiology
- Abstract
In insects, inhibitory neurotransmission is generally associated with members of the cys-loop ligand-gated anion channels, such as the glutamate-gated chloride channel (GluCl), the GABA-gated chloride channels (GABACl), and the histamine-gated chloride channels (HisCl). These ionotropic receptors are considered established target sites for the development of insecticides, and therefore it is necessary to obtain a better insight in their distribution, structure, and functional properties. Here, by combining electrophysiology and molecular biology techniques, we identified and characterized GluCl, GABACl, and HisCl in dorsal unpaired median (DUM) neurons of Locust migratoria. In whole cell patch-clamp recordings, application of glutamate, GABA, or histamine induced rapidly activating ionic currents. GluCls were sensitive to ibotenic acid and blocked by picrotoxin and fipronil. The pharmacological profile of the L. migratoria GABACl fitted neither the vertebrate GABA(A) nor GABA(C) receptor and was similar to the properties of the cloned Drosophila melanogaster GABA receptor subunit (Rdl). The expression of Rdl-like subunit-containing GABA receptors was shown at the molecular level using RT-PCR. Sequencing analysis indicated that the orthologous GABACl of D. melanogaster CG10357-A is expressed in DUM neurons of L. migratoria. Histamine-induced currents exhibited a fast onset and desensitized completely on continuous application of histamine. In conclusion, within the DUM neurons of L. migratoria, we identified three different cys-loop ligand-gated anion channels that use GABA, glutamate, or histamine as their neurotransmitter.
- Published
- 2010
- Full Text
- View/download PDF
41. Dorsal unpaired median neurons of locusta migratoria express ivermectin- and fipronil-sensitive glutamate-gated chloride channels.
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Janssen D, Derst C, Buckinx R, Van den Eynden J, Rigo JM, and Van Kerkhove E
- Subjects
- Algorithms, Amino Acid Sequence, Animals, Chloride Channels drug effects, Chloride Channels genetics, Cloning, Molecular, DNA, Complementary biosynthesis, DNA, Complementary genetics, Data Interpretation, Statistical, Ganglia, Invertebrate cytology, Ganglia, Invertebrate drug effects, Ganglia, Invertebrate physiology, Kinetics, Molecular Sequence Data, Neurons drug effects, Patch-Clamp Techniques, Reverse Transcriptase Polymerase Chain Reaction, Chloride Channels biosynthesis, Glutamic Acid physiology, Insecticides pharmacology, Ion Channel Gating drug effects, Ivermectin pharmacology, Locusta migratoria physiology, Neurons metabolism, Pyrazoles pharmacology
- Abstract
Together with type A GABA and strychnine-sensitive glycine receptors, glutamate-gated chloride channels (GluCl) are members of the Cys-loop family of ionotropic receptors, which mediate fast inhibitory neurotransmission. To date, GluCls are found in invertebrates only and therefore represent potential specific targets for insecticides, such as ivermectin and fipronil. In this study, we identified the functional expression of GluCls in dorsal unpaired median (DUM) neurons of the metathoracic ganglion of Locusta migratoria using electrophysiological and molecular biological techniques. In whole cell patch-clamped DUM neurons, glutamate-induced changes in both their membrane potentials (current-clamp) and currents (voltage-clamp) were dependent on the chloride equilibrium potential. On continuous application of glutamate, the glutamate-elicited current response became rapidly and completely desensitized. Application of glutamate in the presence of 10 microM fipronil or 100 microM picrotoxin reversibly decreased GluCl-mediated currents by 87 and 39%, respectively. Furthermore, 1 microM ivermectin induced a persistent chloride current, suggesting the expression of ivermectin-sensitive GluCl alpha subunits. A degenerate PCR/RACE strategy was used to clone the full-length L. migratoria LmGlClalpha subunit. Finally, RT-PCR experiments demonstrated the presence of LmGluClalpha transcripts in locust DUM neurons. Our results provide the first direct evidence of a functional ivermectin-sensitive GluCl channel on the cell surface of DUM neurons of L. migratoria.
- Published
- 2007
- Full Text
- View/download PDF
42. Pharmacological comparison of two different insect models using the scorpion alpha-like toxin BmK M1 from Buthus martensii Karsch.
- Author
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Bosmans F, Brône B, Sun YM, Zhu RH, Xiong YM, Wang DC, Van Kerkhove E, and Tytgat J
- Subjects
- Animals, Cell Culture Techniques, Electrophysiology, Insect Proteins, Mutation, Neurons drug effects, Species Specificity, Xenopus laevis, Drosophila melanogaster physiology, Locusta migratoria physiology, Neurotoxins pharmacology, Scorpion Venoms pharmacology, Sodium Channels drug effects
- Abstract
In this study, the effect of the scorpion alpha-like toxin BmK M1 was investigated on isolated DUM neurons from Locusta migratoria and compared with the effect on para/tipE voltage-gated Na(+) channels (VGSC), cloned from Drosophila melanogaster. The two insects display different pharmacological properties regarding alpha-like toxins. Moreover, with the aid of the alpha-like toxin BmK M1 and 5 of its mutants, the importance of aromatic residues for the interaction of the toxin with the VGSC in L. migratoria and D. melanogaster, is shown.
- Published
- 2005
- Full Text
- View/download PDF
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