Pierre Devos

Hepatic accumulation and effects of microcystin-LR on juvenile goldfish Carassius auratus L.

After intraperitoneal injection of microcystin-LR (MC-LR) (125 microg kg(-1) body wt.), the concentration of MC-LR in the liver of juvenile goldfish Carassius auratus (30 g body wt.) was assayed by a modified protein phosphatase inhibition method. A temporary accumulation occurred from 3 to 48 h post-injection, followed by a significant decrease between 48 and 96 h. Under our experimental conditions, contamination by MC-LR did not change ionic homeostasis, as attested by blood osmolality values and gill Na(+)/K(+) ATPase activity. Light microscopy observations revealed lesions and cellular necrosis progression, which was concomitant with an increase in enzyme activity of plasma aspartate aminotransferase (AspAT), alanine aminotransferase (AlaAT) and L-lactate dehydrogenase (LDH) and with a decrease of hepatic glutathione-S-transferase (GST) activity. Structural alterations and enzymatic activity modifications became significant within 24 h post-injection. Recovery of hepatocytes on day 21 after MC-LR injection was evident, together with a decrease in the MC-LR equivalent content of the liver.

Obligatory role of membrane events in the regulatory effect of metformin on the respiratory chain function

From recent findings about the indirect effect of metformin (MET) targeted on the respiratory chain complex I, we reconsidered this question and tried to determine the causality of any alteration at this enzymatic level using Xenopus laevis oocytes. Addition of MET (50 microM) reduced by 40% the rotenone-sensitive activity of complex I only in incubating intact oocytes but not in mitochondria isolated by differential centrifugation. The drug prior injected inside these cells had also no measurable effect. In spite of this and the weak binding of MET to the mitochondrial fraction, there was a fairly good correlation between the marked inhibitory action of MET on complex I and its progressive appearance within the oocyte cytoplasm. The intriguing observation that MET as a liposomal form was again able to exert its role when added directly to isolated mitochondria is in accordance with a membrane-mediated uptake and vesicular routing of MET. Furthermore, a temperature-dependent effect was clearly shown. At 4 degrees, oocytes failed to take up efficiently MET and accordingly its subsequent action on respiration was therefore lost. Likewise, MET transport was hindered and inhibition of complex I totally disappeared when a structural analog, asymmetrical dimethylarginine (ADMA), was placed together with MET either at an identical concentration or in excess. These data strongly support the view that MET may recognise some specific membranous sites, likely belonging to effector systems, before penetrating the cell in a bound state via an obscure endocytotic event which still has to be identified.

Dopamine as a Modulator of Ionic Transport and Na+/k+-atpase Activity in the Gills of the Chinese Crab Eriocheir Sinensis

ABSTRACT Dopamine and dibutyryl cAMP induced a significant increase of 22Na+ influx in salt-transporting posterior gills isolated from the Chinese crab Eriocheir sinensis acclimated to fresh water. When isolated gills were incubated and perfused in Cl--free saline (Cl replaced by gluconate), dopamine or dibutyryl cAMP still produced a significant increase of 22Na+ influx. After 1-h perfusion, dopamine increased intracellular cAMP content and Na+/K+-ATPase activity as compared with control perfusions without dopamine.

Cytochrome c oxidase and Na+-K+ ATPase activities in the anterior and posterior gills of the shore crab Carcinus maenas l. After adaptation to various salinities

Abstract 1. 1. There is an increase of the Na+-K+ ATPase activity in the gills of Carcinus maenas adapted to diluted sea-water. 2. 2. A concomitant increase of teh cytochrome c oxidase activity indicates the important part taken by mitochondria for this adaptation.

Effects of atrazine on osmoregulation in the Chinese mitten crab, Eriocheir sinensis

This study integrates results from acute contamination with atrazine of isolated perfused gills, and from in vivo chronic contamination of euryhaline Chinese mitten crabs, Eriocheir sinensis, acclimated to freshwater. Atrazine 1 mg/l in contact with the basolateral membrane (IN) increases the transepithelial potential difference (TEP) from -20.8 +/- 4.9 to -29.7 +/- 3.8 mV in isolated perfused posterior gills (P < 0.01). This effect is only partially explained by a modification of Na(+) and Cl(-) active influxes. No TEP modification is detected when atrazine is added (OUT) indicating that molecular mechanisms located on the basolateral membrane are likely to be the only ones affected. Another explanation would be that cuticular barrier prevents atrazine penetration into the gill. Haemolymph osmolarity, Na(+) and Cl(-) concentrations of crabs living in freshwater contaminated with atrazine 1 mg/l during 14 days are not significantly modified. We conclude that although atrazine can disturb osmoregulatory mechanisms of isolated gills, this pollutant would be of minor importance in affecting osmoregulatory capacities of the Chinese mitten crab in natural conditions.

Potentiating effect of metformin on insulin-induced glucose uptake and glycogen metabolism within Xenopus oocytes

SummaryXenopus laevis oocytes were chosen as the in vitro model for this study with the aim of reconsidering metformin action on the main insulin-responsive glucose pathway. Metformin alone, when present at a therapeutic dose (20 μmol/l) in the incubation medium, did not alter the basal rate of glucose uptake or of glycogen synthesis as measured by [U-14C] D-glucose incorporation. The drug had no effect on the main rate-limiting enzyme implicated in this pathway, i. e. glycogen synthase. In contrast, when combined with 2 μmol/l insulin, metformin led to a specific rise of both free and stored glucose, by 42.4 and 102.3 % respectively. Moreoever, a short-term preincubation of mature oocytes with metformin, but in the absence of glucose, enhanced significantly the amount of synthase a when stimulated by 50 nmol/l insulin (basal 17.4 ± 5.7 %, metformin 21.3 ± 4.1 %, insulin 31.2 ± 4.6 %, metformin together with insulin 62.7 ± 4.2 %, p < 0.005, n = 5). Interestingly, the microinjection of this biguanide, at a final concentration of 20 nmol/l, allowed a similar biochemical response. These data clearly suggest that metformin could act primarily at postreceptor steps which are thought to be key sites in controlling the cellular glucose homeostasis. [Diabetologia (1998) 41: 2–8]

Dopamine D1 receptors in the gills of Chinese crab Eriocheir sinensis.

Our investigations indicate the existence of binding sites for [3H]SCH23390 on crude membrane preparations in the anterior and posterior gills of the Chinese crab, Eriocheir sinensis acclimated to freshwater (FW) or seawater (SW). Maximum specific binding in the posterior gills is always higher than in the anterior gills, independent of saline acclimation. Kd values are similar in the two regions, suggesting the same affinity of both types of gills for the ligand, either from FW or from SW crabs.

Metformin interaction with insulin-regulated glucose uptake, using the Xenopus laevis oocyte model expressing the mammalian transporter GLUT4

The primary goal of this work was to better define, in molecular terms, the impact of metformin on hexose carriers. The methodology consisted of determining the zero-trans kinetics of 2-deoxy-D-glucose uptake for the mammalian insulin-sensitive glucose transporter (GLUT4) expressed in Xenopus laevis oocytes. These cells possessed the specialized protein and, when treated with insulin (2 microM) plus metformin (20 microM), showed a markedly enhanced hexose transport activity (2.4-fold increase over basal) as compared to that of cells incubated in the presence of insulin alone (1.8-fold increase over basal). Kinetic analysis of this process revealed that insulin induced a similar response to that observed for the native carrier, i.e., a higher Vmax. When metformin was added together with insulin, we mainly recorded a significant decrease in apparent Km for the sugar transported, Vmax being only marginally modified. Parathyroid hormone (PTH), which is known to impair the intrinsic activity of GLUT4, prevented the stimulatory effect of metformin in both kinds of oocytes whereas cytochalasin D, which interferes with the translocation of carriers, was without effect. These results suggest that metformin combined with insulin can maintain glucose homeostasis by increasing the catalytic activity of some hexose carriers or by improving the affinity of GLUT4 for glucose.

ACTIVE ABSORPTION OF CL– AND NA+ IN POSTERIOR GILLS OF CHINESE CRAB ERIOCHEIR SINENSIS: MODULATION BY DOPAMINE AND cAMP

Abstract Isolated posterior gills from the Chinese crab Eriocheir sinensis acclimated to freshwater (FW) were used for perfusion. Dopamine and dibutyryl cAMP (db-cAMP) induced a significant increase of 36Cl– influx. When amiloride, a specific sodium selective channels blocker, was added to the incubation saline, it induced a dramatic decrease of 22Na+ influx, and such an effect could be reversed by dopamine or db-cAMP. In the same experimental condition, we observed that ouabain added to the serosal side induced a marked decrease of the stimulating effect of db-cAMP on 22Na+ uptake across the epithelium to the hemolymph, suggesting that dopamine stimulates Na+ and Cl– influxes via cAMP and Na+/K+-ATPase pathways.