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Biomedical subjects

G Flik

Publications and source records attributed to G Flik.

At least 19 recordsLinked to original sources

Na(+) and Ca(2+) pumps in the gills, epipodites and branchiostegites of the european lobster Homarus gammarus: effects of dilute sea water.

Crude homogenates and plasma-membrane-enriched fractions were prepared from the epithelium of the gills, epipodites and branchiostegites of intermoult European lobsters Homarus gammarus, and Na(+)/K(+)-ATPase, Ca(2+)-ATPase and Na(+)/Ca(2+) exchange activities were quantified in these tissues. Lobsters were kept in sea water (salinity 35 ) or were adapted to dilute sea water (22.1 ). The lobster hyperregulates haemolymph osmolarity and Ca(2+) levels in both media. Homogenates of the podobranchs, arthrobranchs and pleurobranchs had comparable Na(+)/K(+)-ATPase specific activities, and mean activities increased significantly for all three types of gills when the animals were kept in dilute sea water. In the epipodites and branchiostegites, Na(+)/K(+)-ATPase specific activities exceeded those in the gills, and exposure to dilute sea water greatly enhanced these activities. In sea water, 80 % of the total Na(+)/K(+)-ATPase activity is associated with the gills and epipodites (each tissue containing 40 %) and 20 % with the branchiostegites; in dilute sea water, the gills contained approximately 25 %, the epipodites 40 % and the branchiostegites approximately 35 % of the total activity, indicating the relative importance of the epipodites and branchiostegites for ionic hyperregulation in dilute media. In plasma membrane vesicles isolated from the gills, epipodites and branchiostegites, Ca(2+) transport driven by ATP and by a Na(+ )gradient was demonstrated. Exposure to dilute sea water enhanced Na(+)/Ca(2+ )exchange and Ca(2+)-ATPase activities in the epipodites and branchiostegites; in the gills, however, Ca(2+) transport activities decreased. The role of these tissues and enzymes in Na(+) and Ca(2+) handling by the lobster is discussed.

Animals

Na(+)/K(+)-ATPase immunoreactivity in branchial chloride cells of Oreochromis mossambicus exposed to copper.

Chloride cells were identified by Na(+)/K(+)-ATPase immunocytochemistry at the light and electron microscope levels in gills of freshwater tilapia Oreochromis mossambicus. Turnover of chloride cells was enhanced by exposing the fish to waterborne copper (3.2 micromol l(-)(1)) for 14 days, as indicated by a 38 % increase in cells expressing proliferating cell nuclear antigen (PCNA) relative to controls. The expression of PCNA was most marked in the central area of the filamental epithelium, from where the chloride cells are thought to originate and migrate. In control fish, chloride cells were associated exclusively with the filamental epithelium. In both controls and copper-exposed fish, two chloride cell populations were seen after Na(+)/K(+)-ATPase immunostaining. These probably represent subpopulations of newly emerged chloride cells: (1) strongly stained cells (mature chloride cells) in the filamental and lamellar epithelium and (2) weakly stained cells, identified by electron microscopy as apoptotic and necrotic chloride cells, mainly in the filamental epithelium. Absolute numbers of mature chloride cells fell, while necrotic and apoptotic chloride cell numbers increased, in copper-exposed fish. A strong correlation could be established for gill Na(+)/K(+)-ATPase specific activity and the number of strongly stained chloride cells in controls and copper-exposed fish and for Na(+)/K(+)-ATPase specific activity and total numbers of immunoreactive cells in copper-exposed fish owing to an increased incidence of weakly staining cells.

Animals

Ultrastructure and distribution dynamics of chloride cells in tilapia larvae in fresh water and sea water.

Integumental and branchial chloride cells of tilapia larvae (Oreochromis mossambicus) were studied at the light-microscopical and ultrastructural level. Total numbers and distribution of chloride cells were quantified after immunostaining of cross sections of the entire larvae with an antibody against the alpha-subunit of Na+/K+-ATPase. The majority (66%) of Na+/K+-ATPase-immunoreactive (ir) cells, i.e. chloride cells, of freshwater tilapia larvae were located extrabranchially up to 48 h after hatching. Five days after hatching, the majority (80%) of chloride cells were found in the buccal cavity. Transfer of 24-h-old larvae to 20% sea water speeded up this process; 24 h after transfer (i.e. 48 h after hatching), the majority (59%) of chloride cells were located in the buccal cavity. The branchial chloride cell population of 24-h- and 120-h-old larvae consisted of immature, mature, apoptotic and necrotic chloride cells. However, relatively more immature chloride cells were observed in freshwater larvae (42-63%) than in (previously studied) freshwater adults (21%), illustrating the developmental state of the gills. After transfer to sea water, the incidence of degenerative chloride cells did not change. Furthermore, the incidence of immature cells had decreased and a new subtype of chloride cells, the "mitochondria-poor" cells, appeared more frequently. These mitochondria-poor chloride cells were characterised by an abundant tubular system and relatively few mitochondria, which were aligned at the border or concentrated in one part of the cytoplasm. Most of these cells did not contact the water. The function of their enhanced appearance after seawater transfer is unknown.

Aging

Na+-K+-ATPase and Na+/Ca2+ exchange activities in gills of hyperregulating Carcinus maenas.

Na+-K+-ATPase and Na+/Ca2+ exchange activities were studied in gills of Carcinus maenas in seawater (SW) and after transfer to dilute seawater (DSW). Carcinus hyperregulates its hemolymph osmolarity through active uptake of Na+, Cl-, and Ca2+. In DSW total Na+-K+-ATPase activity in posterior gills quadrupled; Na+/Ca2+ exchange specific activity was unaffected, and total activity increased 1.67-fold. Short-circuit current (Isc) in voltage-clamped posterior gill hemilamellae was -181 microA/cm2 in SW and -290 microA/cm2 in DSW and up to 90% ouabain sensitive; conductivity was similar in SW or DSW (42 and 46 mS/cm2, respectively) and representative of a leaky epithelium. The new steady state of hemolymph osmolarity 24 h after DSW transfer was preceded, already 3 h after transfer, by increased Na+-K+-ATPase but not Na+/Ca2+ exchange activity. Western blot analysis indicated that the amount of Na+-K+-ATPase protein had increased 2.1-fold in crabs acclimated 3 wk to DSW; however, 4 h after DSW transfer no difference in the amount of Na+-K+-ATPase protein was observed. After DSW transfer branchial cAMP content decreased. A negative correlation between branchial Na+-K+-ATPase activity and cAMP content points to rapid regulation of Na+-K+-ATPase through cAMP-dependent protein kinase A activity. Ca2+ transport may depend on the high-capacity Na+/Ca2+ exchanger coupled to the versatile sodium pump.

Acclimatization

Metallothionein response in gills of Oreochromis mossambicus exposed to copper in fresh water.

Freshwater Oreochromis mossambicus (tilapia) were exposed to 3.2 micromol/l Cu(NO(3))(2) in the water for up to 80 days, and copper (Cu) and immunoreactive metallothionein (irMT) were localized in the branchial epithelium. Cu was demonstrated in mucous cells (MC), chloride cells (CC), pavement cells (PC), respiratory cells (RC), and basal layer cells (BLC) via autometallography combined with alcian blue staining for MC and Na(+)-K(+)-ATPase immunostaining for CC and, on the basis of their location in the epithelium of PC, RC, and BLC. In control fish (water with Cu concentration </=90 nmol/l) incidentally irMT was observed in the area where progenitor cells of the branchial epithelia reside, as demonstrated by proliferating cell nuclear antigen staining. This was also the area where the first increase irMT expression of the Cu exposure was observed. After 2 days of exposure to Cu, irMT was found in CC and PC. From 5 days on, a pronounced irMT staining was observed in BLC of branchial epithelium, which then appeared to migrate and differentiate into mature CC, PC, and RC. We conclude that MT expression in mature CC, PC, and RC requires exposure to Cu in a earlier stage of development of these cells. Once expression is initiated in undifferentiated cells, MT remains expressed throughout the life cycle of the cell.

Animals

Effects of sea water and stanniectomy on branchial Ca(2+) handling and drinking rate in eel (Anguilla anguilla L.).

We examined the effects of seawater adaptation and extirpation of the Stannius corpuscles on branchial Ca(2+) flows, gill plasma membrane Ca(2+) transporters and drinking rate of European eels, Anguilla anguilla. Transepithelial Ca(2+) inflow in the gills increased 2 weeks after transfer of the eels from fresh water to sea water and after stanniectomy. Neither of these treatments changed the membrane density or the affinity of the Ca(2+)-extrusion mechanisms (Ca(2+)-ATPase and Na(+)/Ca(2+)-exchanger) in the gill cells, as measured in basolateral plasma membrane vesicles. We conclude that the increase in the Ca(2+)-transporting capacity observed in the gills of fish exposed to the larger transepithelial Ca(2+) fluxes, resulting from exposure to sea water or stanniectomy, involves an increase in number and/or size of the Ca(2+)-transporting cells, but not in the membrane density of Ca(2+) transporters. Branchial Ca(2+) outflow was higher in stanniectomised than in sham-operated fish. Changes in electrochemical driving forces as well as plasma stanniocalcin or teleocalcin levels may be the basis for the observed differences. Stanniectomy enhanced drinking in stanniectomised eels. Drinking was not further affected by transfer to sea water. These observations suggest that the corpuscles of Stannius are involved in the water balance.

Journal Article

In vivo bafilomycin-sensitive Na(+) uptake in young freshwater fish

In vivo treatment with external bafilomycin A(1), a selective inhibitor of V-ATPase H(+) pumps, reduced whole-body Na(+) influx by up to 90 % in young tilapia and 70 % in young carp. The inhibition was rapidly reversible, with whole-body Na(+) influx rebounding to 280 % of pre-treatment values within 20 min of removal from the bafilomycin. This rebound effect is consistent with the prior accumulation of protons during the period when the cells were exposed to bafilomycin. Bafilomycin also inhibited Cl(-) uptake, an effect that was still apparent 30 min after the removal of bafilomycin. These data provide circumstantial evidence for previous suggestions that Na(+) uptake in freshwater fish is associated with a proton-motive force created by a proton pump and indirect evidence for the major significance of this mechanism in the branchial uptake of Na(+) by freshwater fish.

Journal Article

The stress response of the gilthead sea bream (Sparus aurata L.) to air exposure and confinement.

We investigated short-term effects (up to 24 h) of air exposure and confinement, and long-term effects (up to 11 days) of confinement, to elucidate signalling pathways in the stress response of gilthead sea bream Sparus aurata L. Plasma glucose and lactate were taken as indicators of sympathetic activation, and alpha-melanocyte stimulating hormone (alpha-MSH), adrenocorticotrophic hormone (ACTH) and cortisol as indicators of activation of the brain-pituitary-interrenal (BPI) axis. Air exposure for 3 min resulted, within 30 min, in an increase in plasma concentrations of cortisol, alpha-MSH, glucose, lactate, osmolality and plasma Na, Cl and Mg. Plasma ACTH and beta-endorphin and plasma K, Ca and P did not change. We conclude that air exposure mainly activates the brain-sympathetic-chromaffin cell (BSC) axis. In fish confined at a density of 70 kg/m(3) (compared with 4 kg/m(3) in controls), cortisol, ACTH and alpha-MSH increased within 1 h, indicating activation of the BPI axis. Plasma glucose, Na, Cl and Mg increased with an 8 h delay compared with the response to air exposure. No changes in plasma lactate, osmolality, K, Ca and P were observed. Long-term confinement induced a biphasic cortisol response with peaks at 1 h and at 2 and 3 days. A gradual increase in plasma beta-endorphin concentrations peaked at 7 days; the concentration of alpha-MSH increased rapidly within 1 h and then declined to control values 4 days after the onset of confinement. No changes in ACTH were detected. Our data provide evidence that a stressor-specific activation of the BSC and BPI axes may occur in Sparus aurata.

Adrenocorticotropic Hormone

Effect of low ambient mineral concentrations on the accumulation of calcium, magnesium and phosphorus by early life stages of the air-breathing armoured catfish Megalechis personata (Siluriformes: Callichthyidae).

The accumulation of calcium, magnesium and phosphorus was measured during an 8-week period in the early life stages of the air-breathing armoured catfish Megalechis personata acclimated to low-mineral fresh water (0.073 mmol l-1 calcium, 0.015 mmol l-1 magnesium, <0.001 mmol l-1 phosphate) and high-mineral fresh water (0.59 mmol l-1 calcium, 1.94 mmol l-1 magnesium, <0.001 mmol l-1 phosphate). The fish accumulated calcium twice as fast and phosphorus 1.5 times as fast in low-mineral fresh water (LMF) as in high-mineral fresh water (HMF), while the rate of accumulation of magnesium did not differ in LMF and HMF. The difference in the rates of accumulation of calcium and phosphorus between LMF and HMF was independent of the growth performance (food intake) in LMF and HMF. The mineral content of young M. personata from natural swamps and rainforest creeks in Suriname followed the LMF accumulation curves. The transition from aquatic respiration to bimodal respiration in the third week after hatching did not affect rates of mineral accumulation. The high rates of accumulation of calcium and magnesium of M. personata in LMF of 654 and 58 micromol h-1 kg-1, respectively, exceed the rates of uptake of calcium and magnesium of teleosts reported in the literature. The high rates of mineral accumulation in the early life stages of M. personata reflect the exponential growth during the first 8 weeks after hatching and the requirements of the juveniles while building their dermal armour. M. personata is well-adapted to neotropical fresh waters with an extremely low mineral content. The accumulation of calcium and phosphorus is discussed in relation to the function of the bony armour of M. personata.

Animals

Cloning and expression of two proopiomelanocortin mRNAs in the common carp (Cyprinus carpio L.).

Proopiomelanocortin (POMC) is the precursor for a number of biologically active peptides such as adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin. It is well known that these peptides are involved in the stress response in fish as well as in mammals. We have cloned two different carp POMC cDNAs called, POMC-I and POMC-II. The nucleotide sequences of 955 bp for POMC-I and 959 bp for POMC-II share 93.5% identity in their cDNAs, and the deduced amino acid sequences (both 222 amino acids) are 91.4% identical. In the ACTH and beta-MSH domain, two amino acid substitutions are found, whereas alpha-MSH and beta-endorphin are identical. For beta-MSH, the serine replacement (in POMC-I) by a glycine (in POMC-II) results in a putative amidation site Pro-X-Gly for POMC-II. We used RT-PCR to show that both POMC mRNAs are expressed in the hypophysis, hypothalamus and other parts of the brain of a single fish. Furthermore, in a phylogenetic tree based on POMC sequences the divergence of carp POMC-I and -II from tetraploid animals (salmon, trout and xenopus) is demonstrated.

Amino Acid Sequence

Plasma calcium and stanniocalcin levels of male tilapia, Oreochromis mossambicus, fed calcium-deficient food and treated with 1,25 dihydroxyvitamin D3.

The vitamin D metabolite 1,25 dihydroxyvitamin D3 (1,25(OH)2D3; calcitriol) was injected ip (5 microg/kg-1 body mass daily) into male tilapia, Oreochromis mossambicus, fed calcium-deficient food. Plasma calcium (total and free) and stanniocalcin levels, as well as calcium contents of vertebral and opercular bone and scales, were determined on days 1, 3, and 5. In the treated fish, total plasma calcium levels increased on days 3 and 5. Plasma-free calcium levels remained unaffected. Plasma stanniocalcin levels increased, indicating a response of the Stannius corpuscles to redress 1, 25(OH)2D3-induced hypercalcemia. The calcium contents of bone, operculum, and scales were unchanged. It is concluded that in fish, which lack parathyroid hormone, 1,25(OH)2D3 is hypercalcemic and its action is independent of dietary calcium.

Animals

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

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

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

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

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

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

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