Biomedical subjects

G Flik

Publications and source records attributed to G Flik.

At least 55 records · Page 3Linked to original sources

PubMed1998

Characterisation of glucocorticoid receptors in peripheral blood leukocytes of Carp, Cyprinus carpio L.

Binding studies with [3H]cortisol revealed the presence of a single class of cortisol-binding sites on carp peripheral blood leukocytes (PBL). These binding sites showed high affinity (Kd of 3.8 nM) and low capacity (490 binding sites per cell), indicative of receptor binding. Affinity for cortisone was 254-fold lower than for cortisol. Affinity for the two synthetic glucocorticoids dexamethasone and triamcinolone acetonide (TA) was 4- and 10-fold higher than for cortisol, respectively. Further evidence for the GR character of the receptor came from results showing that cortisol induced apoptosis, which could be blocked by the glucocorticoid analogue RU486. A single meal of cortisol-containing food elevated plasma cortisol concentrations and decreased GR density in PBL, as measured 3 h later. The percentage of circulating B lymphocytes also decreased. Cortisol-induced redistribution of B lymphocytes from the blood, due to cortisol treatment, may explain the decrease of GR numbers in PBL, although downregulation of available GR remains possible.

Animals↗

PubMed1998

Sodium and calcium balance in Mozambique tilapia, Oreochromis mossambicus, raised at different salinities.

Mozambique tilapia, Oreochromis mossambicus, born and raised in five salinities, viz. (relatively soft) fresh water, 25, 50, 75% and full-strength sea-water, were analyzed for ionoregulatory performance (in particular sodium and calcium handling) and growth. This tilapia regulates its blood serum mineral composition rather effectively; however, in sea-water serum concentrations of sodium, chloride and calcium (in males only) were increased, as was the serum osmolarity. In sea-water, the total body sodium pool was significantly enlarged. With increasing salinity, sodium turnover increased. Serum calcium levels and the total body calcium pool were more strictly controlled than those of sodium. The lowest density of chloride cells in opercular epithelium and the lowest branchial Na+-K+-ATPase activity were observed in 50% sea-water; these values were higher in fish kept in waters of lower or higher salinities. Fish grew more rapidly in brackish water. Fish kept in brackish water appeared to depend on food-related calcium for growth as branchial calcium uptake provides no more than 20% of growth related Ca-accumulation.

Adaptation, Physiological↗

PubMed1998

Fatty acids from the cyanobacterium Microcystis aeruginosa with potent inhibitory effects on fish gill Na+/K+-ATPase activity.

Fatty acids from two strains of the cyanobacterium Microcystis aeruginosa, PCC 7820 (a strain that produces the hepatotoxin microcystin-LR, MC-LR) and CYA 43 (a strain that produces only small quantities of MC-LR), were extracted, partially characterised and tested for their inhibitory effect on the K+-dependent p-nitrophenol phosphatase (pNPPase) activity of tilapia (Oreochromis mossambicus) gill basolateral membrane. Thin-layer chromatography of the lipids from dichloromethane:methanol extracts of M. aeruginosa PCC 7820 and CYA 43, using diethylether:isopropanol:formic acid (100:4.5:2.5) as solvent, yielded five inhibitory products from M. aeruginosa 7820 and six from M. aeruginosa CYA 43. None of these products could be related to MC-LR. The inhibitory behaviour of the products mimics that of a slow, tight-binding inhibitor. The inhibitory activity is removed by incubation of extracts with fatty-acid-free bovine serum albumin (FAF-BSA). However, FAF-BSA only partially reversed the inhibition of K+-dependent pNPPase on fish gills pre-exposed to the extracted products. We conclude that M. aeruginosa strains PCC 7820 and CYA 43 produce fatty acids with potent inhibitory effects on K+-dependent pNPPase. The release of these products following lysis of cyanobacterial blooms may help to explain fish kills through a disturbance of gill functioning.

4-Nitrophenylphosphatase↗

PubMed1998

Confocal laser scanning and electron microscopical studies on osmoregulatory epithelia in the branchial cavity of the lobster homarus gammarus

The adult lobster Homarus gammarus is a weak hyper-regulator at low salinity. The objective of this study was to locate the ion-transporting tissues in the branchial chamber of this species, using electron microscopy and confocal laser scanning microscopy with a fluorescent vital stain for mitochondria, DASPMI, which is widely used to locate mitochondria-rich cells in ion-transporting epithelia of fish. A thick mitochondria-rich epithelium is present on the inner side of the branchiostegite and over the entire surface of the epipodites. Ultrastructural observations confirm that this tissue has features typical of an ion-transporting epithelium. When the lobster is transferred to low salinity, these epithelia undergo marked ultrastructural changes, such as an increase in thickness related to the development of basolateral infoldings, the appearance of numerous vesicles and an increase in height of the apical microvilli. In the gills, the branchial filaments are lined by a thin and poorly differentiated epithelium, containing numerous mitochondria; no significant ultrastructural changes were observed in the gills of animals acclimated to low salinity. In summary, in H. gammarus, no evidence of osmoregulatory structures was found in the gills. Differentiated ion-transporting epithelia are present in the branchial cavity, on the inner side of the branchiostegite and on the epipodites; these organs are probably involved in osmoregulation.

Journal Article↗

PubMed1998

Magnesium transport in freshwater teleosts

The magnesium handling of freshwater teleost fish is discussed, with an emphasis on the role of branchial, intestinal and renal transport. In response to the eminent threat of constant diffusive losses of minerals such as magnesium, freshwater fish have developed efficient mechanisms for magnesium homeostasis. Magnesium losses are overcome by the uptake of magnesium from the food, making the intestine an important route for magnesium uptake. Some evidence suggests that intestinal magnesium uptake in fish is a regulated, cellular process. The ambient water is an additional magnesium source for fish, implicating the gills as a secondary route for magnesium uptake. Certainly, in some species, direct uptake from the water, probably via branchial routes, ameliorates the effects of a low-magnesium diet. The hard tissues, representing over 50 % of the total body magnesium pool, form a reservoir from which magnesium can be recruited to perform its functions in the cellular metabolism of soft tissues such as muscle. In fish, as in terrestrial vertebrates, the balance of a variety of elements becomes disturbed when the magnesium homeostasis of the soft tissues is disrupted. However, fish appear to be less sensitive than terrestrial vertebrates to these perturbations. Magnesium is reabsorbed in the kidneys to minimise losses. For renal cells, part of a cellular pathway has been elucidated that would allow absorptive magnesium transport (a magnesium conductive pathway in renal brush-border membranes). In some euryhaline teleosts, the kidneys appear to switch instantaneously to rapid magnesium secretion upon magnesium loading, a response common to marine fish that are threatened by diffusive magnesium entry. This enigmatic mechanism underlies the capacity of some euryhaline species to acclimate rapidly to sea water. Despite the progress made over the last decade, much of the cellular and molecular basis of magnesium transport in the gills, intestine and kidneys remains obscure. The application of fluorescent, radioactive and molecular probes, some of which have only recently become available, may yield rapid progress in the field of magnesium research.

Journal Article↗

PubMed1998

Ca2+ signals during early lymphocyte activation in carp Cyprinus carpio L.

To measure cellular responses and the involvement of increased cytosolic Ca2+ levels ([Ca2+]i), peripheral blood leukocytes (PBL) of carp were loaded with the fluorescent intracellular Ca2+ indicators Fluo-3 and Fura-2. Responses of lymphocytes to T-cell mitogen (phytohaemagglutinin, PHA), to B-cell mitogen (lipopolysaccharide, LPS) and to immunoglobulin (Ig) cross-linking with a monoclonal antibody to carp Ig were measured using flow cytometry. Both T-cell stimulation by PHA and B-cell stimulation by membrane Ig cross-linking evoked a rapid elevation of [Ca2+]i. B-cell stimulation by LPS was not linked to an increase in [Ca2+]i. As judged by the percentage of reacting cells, it was concluded that all Ig-positive lymphocytes reacted to Ig cross-linking by elevating [Ca2+]i. At the single-cell level, the reactions of Fura-2-loaded cells were followed every 6 s using digital imaging microscopy. Both cells displaying spontaneous [Ca2+]i oscillations and non-oscillating cells responded to stimulation with an increase in [Ca2+]i, sometimes, in already oscillating cells, accompanied by an increase in frequency and/or amplitude of the oscillations. These results show that intracellular Ca2+ responses of PBL upon activation resemble those in mammals and form a powerful tool for studies into cell-specific regulation.

Animals↗

PubMed1998

Differential expression of two pro-opiomelanocortin mRNAs during temperature stress in common carp (Cyprinus carpio L.).

Pro-opiomelanocortin (POMC) is the precursor of a number of biologically active peptides, including adrenocorticotropic hormone, alpha-melanocyte-stimulating hormone and beta-endorphin, which are released by the pituitary glands of fish as well as mammals. To quantify the levels of expression of the two POMC mRNAs relative to one another during the response of the common carp to temperature-induced stress, we used reverse transcriptase PCR combined with capillary electrophoresis and laser-induced fluorescence detection. The ratio of POMC-I mRNA to POMC-II mRNA determined in wild-type and four isogenic carp strains was found to be strain-dependent and influenced by temperature. In strain E20xR8, the ratio had altered in favour of POMC-I from 1:3.2 (POMC-I:POMC-II) in fish adapted to 24 degreesC to 1:1.2 in fish adapted to a decrease of 9 degreesC in ambient temperature. A rapid drop in temperature from 24 to 15 degreesC decreased the POMC mRNA ratio at the expense of POMC-I from 1:1.9 in the control fish (strain E4xR3R8) to 1:4.2 3 h after the temperature drop of 9 degreesC. We conclude that both POMC genes are expressed in the common carp and that their expression ratio is strain-dependent and changes in response to ambient temperature.

Adaptation, Physiological↗

PubMed1998

Cellular magnesium transport in the vertebrate intestine.

Magnesium (Mg) absorption across the intestinal epithelium is crucial for Mg homeostasis in all vertebrates. Besides paracellular transport, it involves a cell-mediated component, and this predicts the presence of specific Mg carriers at both the apical and basolateral pole of the epithelial cells. Although the mechanism of transmembrane Mg transport in enterocytes, as in most cell types, has remained an elusive topic for many years, recent studies have provided promising new insights. We here recapitulate the progress that has been made in this field, and advance evidence for membrane carriers that are involved in transcellular Mg transport in the intestine.

Animals↗

PubMed1997

Evidence for P2-purinoceptor-mediated uptake of Ca2+ across a fish (Oreochromis mossambicus) intestinal brush border membrane.

We have studied the effect of ATP on Ca2+ uptake in intestinal brush border membrane vesicles (BBMVs) of the teleost tilapia (Oreochromis mossambicus). ATP stimulated Ca2+ uptake 12-fold over the control, with a linear time course. Ionomycin and detergent treatment did not reduce BBMVs' Ca2+ content, indicating the binding of Ca2+ to a membrane component. A rank order of ATP > ADP > AMP was established for the stimulation of Ca2+ uptake. Adenosine, vanadate, adenosine 5'-[alpha, beta-methylene]triphosphate (a P2x purinoceptor agonist) and adenosine 5'-[gamma-thio]triphosphate (a P-type ATPase inhibitor) were without effect. 2-Methylthioadenosine 5'-triphosphate, a P2y purinoceptor agonist, mimicked the stimulation by ATP. As judged from a kinetic comparison, ATP hydrolysis and the stimulation by ATP of Ca2+ uptake were not compatible. The P2 purinoceptor antagonist suramin and the P2y purinoceptor antagonist Reactive Blue-2 inhibited the Ca2+ uptake stimulated by 1 mM ATP (IC50 0.17 mM and 58 microM respectively). We conclude that ATP-stimulated Ca2+ uptake in tilapia intestine is dissociated from ATPase activity and is mediated through a P2 purinoceptor.

Adenosine Triphosphatases↗

PubMed1997

Conservation of apoptosis as an immune regulatory mechanism: effects of cortisol and cortisone on carp lymphocytes.

This is the first study to show that apoptosis as an immune regulatory mechanism is conserved in fish, demonstrating its importance in maintaining immunological homeostasis. The data further show that this mechanism is subject to control by glucocorticosteroids. Carp plasma cortisol concentrations increase from 20 to 434 ng/ml and cortisone from 5 to 50 ng/ml within 9 min of the onset of handling stress. At basal steroid concentrations in vitro, cortisol, but not its conversion product cortisone, inhibits proliferation of peripheral blood lymphocytes (PBL), as measured by [3H]thymidine incorporation. Induction of apoptosis in activated PBL is the apparent mechanism of cortisol action. In nonstimulated PBL cultures, apoptosis is induced by neglect (a lack of stimulating signals). Stimulation with LPS or PHA rescues lymphocytes from this type of apoptosis. Stimulated PBL populations, however, are sensitive to cortisol-induced apoptosis. Culture supernatants from activated PBL protect PBL from apoptosis by neglect, probably by supplying a growth signal. These supernatants, however, have no effect on cortisol-induced apoptosis.

Animals↗

PubMed1997

Acid stress induces a D1-like dopamine receptor in pituitary MSH cells of Oreochromis mossambicus.

A 7-day exposure of tilapia (Oreochromis mossambicus) to water with a pH of 4.5 activates their pituitary melanophore-stimulating hormone (MSH) cells to preferentially release diacetyl alpha-MSH as an important corticotrope (13). We here focus on the control of alpha-MSH release by dopamine in tilapia exposed to water with low pH ("low-pH tilapia"). The MSH cells of low-pH tilapia showed a decreased sensitivity to inhibitory concentrations (10(-7)-10(-5) M) of dopamine compared with controls. Low concentrations (10(-14)-10(-8) M) of dopamine stimulated the release of alpha-MSH in low-pH tilapia but not in controls. Strong pharmacological evidence for a stimulatory dopamine receptor (D1-like) was obtained: the D1-agonists SKF-38393 and 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazep ine hydrobromide (6-chloro APB) had a stimulatory effect on the release of alpha-MSH in low-pH tilapia MSH cells but not in controls. The selective D2-agonists quinpirole and 2-hydroxy apomorphin inhibited the release of alpha-MSH in controls as well as in low-pH tilapia, and there was no difference in the sensitivity of the cells to these agonists. We conclude that only MSH cells of low-pH exposed tilapia exhibit a D1-like receptor activity. A comparable D2-like receptor activity, as demonstrated by specific D2-receptor agonists, is present in both controls and low-pH-adapted fish. The apparent loss of sensitivity of the MSH cells to inhibitory concentrations of dopamine, therefore, must be caused by the activation of the D1-like receptors and not by changes in the activity of the D2-like receptor proper. Stimulatory concentrations of dopamine not only quantitatively but also qualitatively enhanced the corticotropic activity of the released alpha-MSH, as indicated by the elevated ratio of diacetyl and monoacetyl alpha-MSH. This effect was mimicked by the D1-like agonists SKF-38393 and 6-chloro APB, indicating that the D1-like receptor activity is responsible for the enhancement of the di/mono ratio.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed1997

Mitochondria-rich cells in gills of tilapia (Oreochromis mossambicus) adapted to fresh water or sea water: quantification by confocal laser scanning microscopy

We used confocal laser scanning microscopy to validate a new and fast co-labelling method to study the distribution of mitochondria-rich (MR) cells in gill filaments and to differentiate between MR cells that are in contact with the water (cells labelled with both DASPMI and Concanavalin-A) and those that are not (DASPMI-positive only). This method was used to describe differences in MR cell density that occur in the gills of tilapia Oreochromis mossambicus adapted to fresh water or sea water. In fresh water, the total MR cell density was 6233 cells mm-2 and the density of the subpopulation of MR cells that are in contact with the water was 3458 mm-2. After seawater adaptation, cell density decreased to 3061 cells mm-2 for all MR cells of which 2445 cells mm-2 were in contact with water. The percentage of double-labelled MR cells in the total MR cell population had increased from 55 to 80 %. MR cell size (measured as the maximal cross-sectional area) increased from 87 µm2 in fresh water to 217 µm2 in sea water. Biochemical determination of specific and total Na+/K+-ATPase activity in gill homogenates showed no difference between freshwater- and seawater-adapted fish. Quantification of 'mature' chloride cell density in fixed gill filaments using scanning electron microscopy resulted in an overestimate of chloride cell density due to shrinkage of the sample.

Journal Article↗

PubMed1997

Na+ and Ca2+ homeostatic mechanisms in isolated chloride cells of the teleost Oreochromis mossambicus analysed by confocal laser scanning microscopy

Chloride cells (CCs; recognised by the vital mitochondrial stain DASPEI) and pavement cells (PCs) isolated from tilapia opercular epithelium were adhered to Cell-Tak-coated glass coverslips and loaded with fluorescent probes for the measurement of intracellular concentrations of Na+ or Ca2+. Basal levels of cytosolic Na+ and Ca2+ ranged from 6.4 to 16.5 mmol l-1 and from 76 to 110 nmol l-1, respectively, and did not differ between CCs and PCs. In CCs, inhibition of Na+/K+-ATPase by ouabain or Cu2+ increased intracellular [Na+]. Replacing extracellular Na+ with N-methyl-d-glucamine+ led to a rise in cytosolic [Ca2+] that was dependent on the extracellular [Ca2+], indicating that a Na+/Ca2+ exchanger was operating in reverse mode (importing Ca2+). The forward mode of this exchanger could be demonstrated by inhibition with bepridil. The CC has various pathways for passive Na+ influx: a tetrodotoxin-sensitive pathway, an amiloride-sensitive pathway and other as yet unidentified pathways.

Journal Article↗

PubMed1997

Mg2+ transport in plasma membrane vesicles of renal epithelium of the Mozambique tilapia (Oreochromis mossambicus)

To elucidate the mechanisms involved in Mg2+ transport at the apical and basolateral poles of the renal tubular epithelium, apical and basolateral plasma membrane vesicle preparations were derived from kidney tissue of freshwater- and seawater-adapted Mozambique tilapia Oreochromis mossambicus. Brush-border preparations were enriched 15.8-fold in alkaline phosphatase activity and consisted almost exclusively of right-side-out membrane vesicles. Basolateral membrane preparations were enriched 7.5-fold in Na+/K+-ATPase activity and contained resealed vesicles and leaky membrane fragments. Mg2+ association with brush-border and basolateral plasma membranes, traced using radioactive 27Mg, occurred in an osmotically active space. In all instances, Mg2+ binding to the vesicular membrane was low compared with the vesicular uptake. Mg2+ equilibration across the vesicular membrane of brush-border preparations was rapid and sensitive to the presence of extravesicular Ca2+, suggesting that the apical membrane of the renal epithelium contains a transport pathway for divalent cations. Application of various ionic gradients did not affect vesicular Mg2+ transport in apical and basolateral membrane preparations, suggesting the presence of an ion-coupled transport mechanism. ATP or ATP--S did not stimulate Mg2+ fluxes, indicating that Mg2+ transport does not proceed via an ATP-driven or activated transporter. In these aspects, vesicular Mg2+ transport was similar in seawater and freshwater preparations. These results suggest that the apical membrane of renal epithelial cells lacks an active secretory Mg2+ transport mechanism. We propose that the Mg2+ conductivity of the apical membrane reflects a route for downhill Mg2+ entry and is involved in renal Mg2+ reabsorption.

Journal Article↗

PubMed1996

Cloning and sequence analysis of a hypothalamic cDNA encoding a D1c dopamine receptor in tilapia.

Physiological and pharmacological studies have indicated that during acid stress a D1-like dopamine receptor becomes functional on intermediate pituitary melanocyte-stimulating hormone cells of tilapia (Oreochromis mossambicus). As a first step towards physiological expression studies we isolated a D1-like dopamine receptor from a tilapia hypothalamus cDNA library. Construction of a phylogenetic tree of most of the D1-like receptors known in human, rat, Xenopus, goldfish and Drosophila revealed that the here presented clone is most likely the tilapia equivalent of the Xenopus D1c dopamine receptor.

Amino Acid Sequence↗

PubMed1996

Magnesium transport across the basolateral plasma membrane of the fish enterocyte.

In tilapia (Oreochromis mossambicus) intestine, Mg2+ transport across the epithelium involves a transcellular, Na+- and Na+/K+-ATPase dependent pathway. In our search for the Mg2+ extrusion mechanism of the basolateral compartment of the enterocyte, we could exclude Na+/Mg2+ antiport or ATP-driven transport. Evidence is provided, however, that Mg2+ movement across the membrane is coupled to anion transport. In basolateral plasma membrane vesicles, an inwardly directed Cl- gradient stimulated Mg2+ uptake (as followed with the radionuclide 27Mg) twofold. As Cl--stimulated uptake was inhibited by the detergent saponin and by the ionophore A23187, Mg2+ may be accumulated intravesicularly above chemical equilibrium. Valinomycin did not affect uptake, suggesting that electroneutral symport activity occurred. The involvement of anion coupled transport was further indicated by the inhibition of Mg2+ uptake by the stilbene derivative, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid. Kinetic analyses of the Cl--stimulated Mg2+ uptake yielded a Km (Mg2+) of 6.08 +/- 1.29 mmol . l-1 and a Km (Cl-) of 26.5 +/- 6.5 mmol . l-1, compatible with transport activity at intracellular Mg2+- and Cl--levels. We propose that Mg2+ absorption in the tilapia intestine involves an electrically neutral anion symport mechanism.

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

PubMed1996

Kinetics of Cu2+ inhibition of Na+/K(+)-ATPase.

The interaction of Cu2+ with enzymatic activity of rabbit kidney Na+/K(+)-ATPase was studied in media with buffered, defined free Cu2+ levels. The IC50-values are 0.1 mumol/l for Na+/K(+)-ATPase and 1 mumol/l for K(+)-pNPPase. Dithiothreitol (DTT) reverses the inhibitory effect of Cu2+ in vitro. Cu2+ exerts non-competitive effects on the enzyme with respect to Na+, K+, ATP or pNPP, but has a mixed-type inhibitory effect with respect to Mg2+. It is concluded that the appreciation of the inhibitory effect of Cu2+ on this enzyme requires carefully composed assay media that include a buffer for Cu2+, and that the IC50-values calculated according to this model indicate that Cu2+ may be more toxic than previously anticipated.

4-Nitrophenylphosphatase↗

PubMed1996

The effects of cyanobacteria and the cyanobacterial toxin microcystin-LR on Ca2+ transport and Na+/K+-ATPase in tilapia gills

The effects of cytotoxic substances from cyanobacteria on ionic transport processes in tilapia (Oreochromis mossambicus) were examined. Inhibitory effects on ionic transport including whole-body Ca2+ fluxes and P-type ATPases of the gill were found. The compounds tested were (1) purified microcystin-LR (MC-LR), a heptapeptide hepatotoxin produced by the cyanobacterium Microcystis aeruginosa, (2) extracts from M. aeruginosa strain PCC 7820, a strain producing MC-LR and other microcystin variants, and (3) extracts of M. aeruginosa CYA 43, a strain producing toxins including small quantities of MC-LR. Whole-body Ca2+ influx was inhibited by a 24 h exposure to extracts of M. aeruginosa CYA 43 and 7820, but not by exposure to an equivalent amount (90 mg l-1) of purified MC-LR. Shorter exposure times (4 h) were ineffective. Fish exposed to extracts from M. aeruginosa CYA 43 showed significant plasma hypocalcaemia. Both strains of M. aeruginosa inhibited Ca2+ uptake by basolateral plasma membrane vesicles (BLMVs), endoplasmic reticulum (ER) and mitochondria, as well as BLMV K+-dependent p-nitrophenol phosphatase (pNPPase) activity. The hydrophobic fractions of the cyanobacterial extracts were the most potent, inhibiting BLMV, ER and mitochondrial Ca2+ uptake by up to 99 %, but they were less inhibitory of BLMV K+-dependent pNPPase activity. Purified MC-LR was without effect on these preparations. In conclusion, cytotoxic substances from cyanobacteria have the potential to disrupt normal physiological processes dependent upon Ca2+ transport processes in tilapia gills.

Journal Article↗