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P03-04

REGULATION OF ATP-SENSITIVE K+ CHANNELS BY ACUTE CHANGES IN TEMPERATURE IN FISH CARDIAC MYOCYTES

Paajanen V., Vornanen M.

Sarcolemmal potassium efflux through ATP-sensitive channels (IKATP) is assumed to protect cardiac myocytes in ischemia and hypoxia by reducing the duration of action potential and hence cardiac contractility. The hypoxic opening of IKATP is mediated by the depletion of intracellular ATP which tends to balance ATP supply and demand. Here we show that in ventricular myocytes of the fish heart the opening and closing of IKATP is induced by acute temperature changes.

Using whole-cell, cell-attached and inside-out configurations of the patch-clamp method, we studied IKATP in ventricular cardiac myocytes of an extremely eurythermic and anoxia-tolerant fish species, the crucian carp (Carassius carassius). Under normoxic (O2 = 8.6 mg/L) conditions and in the presence of 5 mM ATP in the pipette solution, IKATP was induced by a gradual rise of temperature above the physiological body temperature of 5°C. Once induced, the amplitude of the IKATP was proportional to the extent of temperature rise (95.3 ± 9.7 pS/pF at 18°C), fully reversible by cooling and inhibited with 10 mM glibenclamide, a blocker of IKATP. The temperature-induced increase of the IKATP was only partly explained by the increase in single channel conductance from 32 pS at 5°C to 50 pS at 18°C (Q10=1.43). Acute temperature changes had no effect on the kinetics of the IKATP in inside-out patches. IKATP of the crucian carp cardiac myocytes were characterized by extremely low sensitivity to inhibition by ATP (ID50= 1.35 mM).

These results indicate that in ventricular myocytes of the crucian carp heart, the opening and closing of IKATP is regulated by acute temperature changes. By this means, the IKATP may modify the duration of ventricular AP and hence regulate cardiac contractility in temperature-dependent manner. The molecular mechanisms by which temperate regulates the opening and closing of the ATP-sensitive K+ channels remains to be shown.

University of Joensuu - FINLAND

P03-05

OXIDATIVE MECHANISM IN THE TOXICITY OF CADMIUM.

Krichah R., Chater S., Ben Rhouma K., Tebourbi O., Favier A., Sakly M.

Cadmium (Cd) an abundant nonessential element is widely used in electroplating and galvanizing. Soluble cadmium salts accumulate and result in toxicity to various tissues: liver, brain, thymus and central nervous system. In the present study we assessed the ability of acute Cd-exposure to induce an oxydatif stress through the determination of the total antioxydant state (TAS, Kit Randox), glutathion peroxidase activity (GPX) and tissue metallothioneins (MTs) levels. Male wistar rats, weighing 100-150g were injected with a single dose of 1.5 or 3mg Cadmium chloride/kg body weight (bw)/(ip). Control animals received the same volume of saline. All animals were sacrified by decapitation 24 or 48 hours later. Cd-administration induced a decrease of the total antioxydant status (TAS) 24 hours later (0.56 ± 0.08 and 0.63 ± 0.06 mmol/l respectively for 1.5 and 3mg Cd/kg bw vs 0.77 ± 0.03 mmol/l ) and a depletion of plasma GPX activity (2.27 103 ± 0.08 and 2.43 103 ± 0.12 vs 5.04 103 ± 0.16 U/l). After 48 hours a partial recovery of this activity was observed (3.19 103 ± 0.06 and 3.52 103 ± 0.04 vs 5.04 103 ± 0.16 U/l ), while cytochrom c reduction was more important in the treated rats thymus. The same treatment stimulated MTs biosynthesis in thymus and liver 24 hours later and this effect was more important after 48 hours. These results indicate that acute Cadmium chloride administration induced an oxidative stress state that may contribute to its toxic effects.

Sciences Faculty of Bizerte, Tunisia and La Tronche Hospital, Grenoble, France

P03-06

DDT CYTOTOXICITY IS NOT MEDIATED BY CORTICOSTEROIDS

Tebourbi O., Ben Rhouma K., Krichah R., Hallegue D., Chater S., Sakly M.

DDT (1,1 bis (p-chlorophenyl) 2,2,2 trichloroethane)is an organochlorine insecticide. DDT is very neurotoxic, hepatotoxic and has an endocrine effect. However, the mechanism of action of this pesticide is not clear. The purpose of the present study is to evaluate the effect of DDT on adrenal gland,the thymic glucocorticoid receptors and serum corticosterone levels. Male Wistar rats received 50 or 100mg of pesticide/kg b.wt. (i.p) during ten consecutive days. After adrenalectomy, the relative weight of the thymus increased confirming the thymolytic effect of glucocoticoids. Administration of DDT induced a thymic atrophy in both normal and adrenalectomized rats. The adrenalectomy also led to an hepatic atrophy. But, after subacute exposure of DDT, the liver weight increased significantly in normal and in adrenalectomized animals. This data associated to the unchanged relative weight of adrenal gland in DDT-injected rats suggest that the pesticide has a direct toxic effect indepentamently of the corticotropin axis. The normal architecture was seen in rats treated with 50mg of DDT while those treated with 100mg of pesticide developped necrosis and cytoplasmic vacuolization in the reticularis and fasciculata zona. The density of glucocorticoid receptors treated with 100mg/kg of DDT increased slightly (552.8 ± 5.1 vs 456.2 ± 4.9fmol/mg protein). Serum corticosterone levels were decreased with the dose of 100mg. This decrease may explain the elevation of thymic glucocorticoid receptor density induced by the higher dose of pesticide. These results suggest that DDT exerted cytotoxicity action directly on somatic cells and didn’t activate the adrenal secretion. Nevertheless, the high dose of pesticide decreased the secretion of corticosteroids probably by inhibiting gland streoidogenesis pathways.

Sciences Faculty of Bizerte, Tunisia

P03-07

INCREASED EXPRESSION OF THE IKR IN CARDIAC MYOCYTES OF THE COLD-ACCLIMATED TROUT

Vornanen M.

Delayed rectifier K currents provide major repolarising power during the phase 3 of cardiac action potential (AP). To prevent temperature-dependent changes in AP duration and cardiac excitability, seasonal temperature adaptation of the heart in ectothermic animals is expected to involve modification of K currents. Hence we studied the effects of thermal acclimation on the rapid component of the delayed rectifier current, IKr, in atrial myocytes of the trout heart. The density of the IKr at 0 mV was 12.7±1.3 and 13.1±2.9 pA pF-1 for cold-acclimated (CA, 4°C) and warm-acclimated (WA, 18°C) trout, respectively, indicating an almost perfect thermal compensation of the current amplitude. On the other hand, deactivation and inactivation kinetics of the IKr were not changed by temperature acclimation, even though they were strongly affected by acute temperature changes. To address the physiological importance of the IKr, currents were elicited by using physiological APs as the command waveform. In agreement with the square wave stimuli, the densities of the IKr were equal for CA trout at 4°C and WA trout at 18°C. More surprisingly and in apparent conflict with the strong temperature-dependency of the IKr, the activation of the IKr was much faster in CA trout at 4°C than in WA trout at 18°C. This is, however, explained by the slow deactivation kinetics of the IKr in the cold as it prevents the closing of the activation gate at physiological heart rates (40/min) at 4°C. The open (non-deactivated) channels generate an immediate outward current when driving force is restored by the depolarisation of the next AP. In conclusion, incomplete deactivation of the channels at low temperatures and cold-induced increase in the density of the IKr generate fast and large repolarising currents in atrial myocytes of CA trout. These properties of the IKr maintain the duration of AP short for adequate cardiac function in the cold environment.

University of Joensuu, Department of Biology, P.O. Box 111, 80101 Joensuu, Finland

P03-09

MECHANISMS OF AMINO ACID TRANSPORT BY THE ISOLATED INTESTINE OF THE FROG RANA ESCULENTA

Saïdane D.

This work was aimed at studying the mechanisms of intestinal transport of aminoacids in consideration of their interactions with the transport of major inorganic ions. The study was performed on the isolated intestine of the green frog Rana esculenta by using in vitro approaches: Ussing chambers, membrane vesicles from apical and basolateral membranes of enterocytes, apical short-term uptake of labelled solute (Schultz, Curran, Chez and Fuisz, 1967).

Combination of these techniques allowed us to characterize the following transport mechanisms for aminoacids that we defined by analogy with systems already described in mammals and in certain fish :

1- A neutral aminoacid transporter close to the B (previously NBB) system

2- A Phenylalanine system transferring long chain aminoacids (phenylalanine and methionine)

3- A Chloride-dependent transporter for methionine and phenylalanine, like the mammalian Bo,+ system

4- A Glycine system sensitive to chloride, comparing to the Gly one of mammals

5- A Sodium-dependent mechanism carrying alanine and serine, equivalent to the ASC system

6- Another sodium-dependent transferring lysine and possibly all basic aminoacids, reminiscent of the Y+ system of the rat

7- A Sodium-independent system for the uptake of phenylalanine and very similar to the L system which ensures the sodium-dependent transport of long chain neutral aminoacids

8- Another sodium-independent system for alanine, sharing these properties with the b system described in mammals, now called b0,+

Acknowledgements : I wish to thank Dr B. Lahlou and Dr J. Ehrenfeld for helpful advice

Faculté de Pharmacie, Monastir, Tunisie

P03-10

NITRIC OXIDE IN EXPERIMENTAL HEPATIC FIBROSIS

Parvu A.E., Parvu M., Plesca-Manea L., Hoteiuc O.

The role of nitric oxide (NO) in the pathogenesis of liver diseases has been extensively studied. Hepatocellular damages initiate an inflammatory reaction. Elevated nitric oxide production is assumed to be responsible for the pathological changes in many inflammatory conditions.

The purpose of this study was to assess the effects of the chronic stimulation or inhibition of nitric oxide synthesis in male Wistar rats with CCl4-induced hepatic fibrosis. Plasma levels of production of citrulline from arginine (Boyde method), nitrite and nitrate (Griess reaction) were determined in rats before and after intraperitonealy administration of L-arginine (L-ARG), NG-nitro-L-arginine methyl ester (L-NAME), pentoxifiline, dexametasone and methilen blue. We analyzed the relationships between the levels of NO synthesis and some hepatic parameters (ASAT, ALAT, LDH, gamma-GT, AP, bilirubin0. the resulta showed that animals with toxic hepatic fibrosis had correlated elevation of plasma citrulline and nitrite/nitrate levels, most due to an increase stimulation of the iNOS and a lower stimulation of the cNOS types. The hepatic parameters had a parallel increase with the induced form of NO.

CONCLUSION:The most important source of NO in hepatic fibrosis is the induced one. Citrulline and nitrite/nitrate levels may indicate the rate of hepatic damage as do other plasmatic parameters.

University Of Medicine And Pharmacy "IULIU HATIEGANU", CLUJ-NAPOCA, ROMANIA

P03-11

APPLICATION OF INHALATED PHOSPHOLIPID LIPOSOMES IN HCL-INDUCED LUNG INJURY

Steneva J., Jordanova A., Lalchev Z., Ninio S., Neicheva T., Petkova D.

The aim of this study was to evaluate the inhalatory application of phosphatidylcholine multilayer liposomes (PL) in HCl – induced acute respiratory distress sindrome (ARDS) in rabbits.

ARDS was induced by administration of 0.2 N HCl via intratracheal instillation for 45 min. After induced ARDS animals under artificial lung ventilation were retreated with PL in saline (25mg/kg body weight) for 60 min. Arterial blood gas (ABG) analysis was performed at 15, 30, 45 and 60 min after PL application. Untreated animals were ventilated for the same time during all the experiments and ABG analysis was performed as well. Animals were killed with thiopental and bronhoalveolar lavage fluid (BALF) was investigated for phospholipids, cholesterol and specific surfactant protein content. The equilibrium surface tension of monolayers obtained from BALF was determined by Wilhelmy balance.

HCl- lung injury caused decrease of arterial PO2 to the fraction of inspired O2 (PaO2/FiO2) ratio more than 50% compared to the control. We obtained high respiratory acidosis – increase of arterial CO2 ( PaCO2 ) and decrease of blood pH. An increase of alveolae – arterial PO2 (A-a pO2 ) was also detected. The inhalation of PL led to reversion of gas exchange even at 30 min after application, saturation of arterial blood, decrease of A-a pO2. Changes in blood pH we obtained at 45 min after the application of PL and at 60 min pH value returned to the control value. HCl- lung injury caused significantly increase of total protein and cholesterol content, decrease of total phospholipids and percent participation of phosphatidylcholine (PC) and increase of that of sphingomyeline in BALF compared to the control. These changes correlated with biophysical parameters. The sample surface tension was decreased. The application of PL led to reverse of the percent participation of PC , biophysical parameters to the control value and lung function as well.

Institute of Biophysics, Bulgarian Academy of Sciences, Sofia, Bulgaria

P03-12

EFFECT OF EXPOSURE TO MAGNETIC FIELD ON SOME BLOOD PARAMETERS IN MALE RATS

Amara S., Abdelmelek H., Ben Rhouma K., Sakly M.

The present work was undertaken in order to investigate the effects of magnetic field (MF) on hematopoiesis, serum lactate dehydrogenase and transaminases activities in male rats. The exposition of rats 1hour/day for 5 consecutive days to MF of 128 mT (m tesla) had a weak effect on hemoglobin and hematocrite levels. The same treatment increased significantly serum lactate dehydrogenase activitie, whereas alanine aminotransferase and aspartate aminotransferase activities remained unchanged. MF exposure for 30 consecutive days increased significantly hemoglobin, the red , the white blood cells and the platelet number. It was concluded that MF expopsure might induce hemoglobin conformational change causing an hypoxia-like state that stimulated hematopoiesis. Sub-chronic exposure to MF increased also serum lactate dehydrogenase and transaminases activites suggesting hepatic damage.

Key words: Magnetic field, LDH, transaminases, hematopoiesis, rat.

Faculté des Sciences de Bizerte, Jarzouna, Tunisia

P03-13

LOCUS COERULEUS MODULATES THE HYPOXIC VENTILATORY RESPONSE IN ADULT RATS

Soulage C., Perrin D., Cottet-Emard J.M., Pequignot J.M.

Upon exposure to hypoxia, the initial and most important response is an increase in alveolar ventilation. There is now growing evidence that the medullary catecholaminergic cell groups (A1C1, A2C2, A5, A6) participate in the ventilatory response to hypoxia. The A6 cell group, also referred to as locus coeruleus (LC), is the largest cluster of noradrenergic cell bodies of the brain. The present study was designed to assess the involvement of A6 noradrenergic cell group, for the establishment of the ventilatory response to short-term hypoxia. The breathing response to acute hypoxia (10%O2) was measured in awake and unrestrained rats by barometric plethysmography 15 days after a unilateral lesion of LC with 6-hydroxydopamine (6-OHDA). The 6-OHDA infused “in situ” caused a major loss of noradrenergic neurones in A6 area assessed in histology and in neurochemistry. The unilateral lesion fails to alter minute ventilation, tidal volume or frequency under basal conditions (room-air, 21%O2). During a 12 minutes hypoxic challenge (10%O2) whereas minute ventilation remains unaffected, 6-OHDA treated rats exhibit a lower tidal volume (-67%) than sham-operated ones. This blunted response in tidal volume is however counter-balanced by an enlarged response in frequency. Our results strongly suggest that separated mechanisms and distinct structures are acting in the regulation of tidal volume and respiratory frequency. We concluded that i) LC noradrenergic neurones are not essential for breathing modulation under normoxia, ii) noradrenergic neurones of the LC and catecholaminergic mechanisms are involved in regulation of tidal volume under hypoxia.

UMR CNRS 5123 - Physiologie Intégrative, Cellulaire et Moléculaire, Laboratoire de Physiologie de l'Environnement. LYON - FRANCE

P03-14

INVOLVEMENT OF CGRP BUT NOT TACHYKININS IN LOCAL PRESSURE-INDUCED VASODILATION

Merzeau S., Fromy B., Abraham P., Saumet J.L.

Capsaicin-sensitive afferent neurones are connected to cutaneous receptors which enable them to detect noxious stimuli that are potentially or actually harmful to the tissue. Neurokinins and calcitonin gene-related peptide (CGRP) are released from these peripheral nerve terminals following their activation. Local pressure-induced vasodilation (PIV) is a neural vasodilator response to non-nociceptive externally applied pressure in the skin. The first aim of the present study was to determine whether cutaneous PIV exists in rats and is dependent on capsaicin-sensitive fibres as it is in humans. We then examined whether CGRP and neurokinin receptors are involved in this reflex. Cutaneous blood flow was measured by laser Doppler flowmetry during 11.1 Pa.sec-1 increases in local externally applied pressure in untreated anaesthetised rats. The involvement of capsaicin-sensitive fibres in this mechanism was tested in rats treated neonatally with capsaicin. Separate groups of adult rats were treated with CGRP8-37 (100 µg.kg-1, i.v.), SR140333 (200 µg.kg-1, i.v.), SR48968 (4 mg.kg-1, i.v.) or SR142801 (1 mg.kg-1, i.v.) to antagonise CGRP, NK1, NK2 or NK3 receptors, respectively. PIV was found in rats, as in humans. It was abolished by neonatal treatment with capsaicin and intravenous administration of CGRP8-37 but remained unchanged with SR140333, SR48968 and SR142801 treatments compared to their respective vehicles. These results suggest that PIV depends on capsaicin-sensitive fibres in rats, as in humans. Furthermore, it appears that CGRP plays a major role in this capsaicin nerve mediated vasodilation in rat skin, whereas neurokinins appear to have no role in PIV.

Laboratory of Physiology, Medecine school, Rue Haute-de-Reculée, 49045 Angers - FRANCE

P03-15

THE EFFECT OF ISOFLURANE ON THE SKIN PRESSURE-INDUCED VASODILATION

Fizanne L., Fromy B., Preckel M.P., Sigaudo-Roussel D., Saumet J.L.

Since general anesthesia has shown to attenuate endothelium-dependent vasodilation, it was of interest to verify whether general anesthesia would modify a skin vasodilation in response to local pressure application, which is endothelium-dependent. To study the effect of general anesthesia on pressure-induced vasodilation development, we examined the effects of isoflurane in light and deep states. Skin blood flow was measured by laser Doppler flowmetry during 11.1 Pa sec-1 increases in locally applied pressure in anesthetized rats. Rats were treated with low or high doses of isoflurane. Following the administration of low doses of isoflurane, skin blood flow increased from baseline, with increasing local pressure application (+37±10% at 2.0 kPa). The increase in skin blood flow disappeared in treated rats with high doses (-205% at 2.0 kPa), even when the anesthesia-induced hypotension was corrected by gelofusine infusion (-2010% at 2.0 kPa). Whereas sodium nitroprusside-induced vasodilation was developed with low and high doses of isoflurane, acetylcholine-induced vasodilation was impaired with high doses compared to low doses. These data show that pressure-induced vasodilation is abolished with high doses of anesthetic. It is not the anesthesia-induced hypotension, but the depth of anesthesia, which can lead to the disappearance of pressure-induced vasodilation by an alteration of endothelial function.

Lab. of Physiology, Medecine school, Rue Haute-de-Reculée, 49045 Angers, France

P03-16

EDHF INVOLVEMENT IN SKIN PRESSURE-INDUCED VASODILATION

Garry A., Merzeau S., Fromy B., Saumet J.L.

At least three different vasodilator agents are synthesised by the endothelium upon exposure to mechanical forces or to receptor-dependent agonists: nitric oxide (NO), prostaglandins and the endothelium-derived hyperpolarising factor (EDHF). Skin pressure-induced vasodilation (PIV) is a neuronal response to locally applied pressure discovered in humans and in rats. This new mechanism results from a complex response originating from capsaicin-sensitive skin sensory fibres and local secretion NO and prostaglandins. No information has been published on EDHF in this mechanism. The aim of the present study was to examine the EDHF role in the PIV development in treated rats with a combined infusion of charybdotoxin and apamin and in controls. Skin blood flow was measured by laser-Doppler flowmetry in response to a progressive local pressure applied to the skin. In treated rats as in controls, the skin vascular conductance increased with increments of local pressure (56.5±11.1% vs 59.5±8.4%, P>0.05). We report here that the vasodilator capacity was not altered in rats treated with charybdotoxin+apamin compared to controls. In conclusion, our study indicates that when NO pathway is intact, there is no or little implication of EDHF in the cutaneous PIV development in rats.

Laboratory of Physiology, Medecine school, Rue Haute-de-Reculée, 49045 ANGERS - FRANCE

P03-17

CUTANEOUS PRESSURE-INDUCED VASODILATION IS DEPENDENT ON ENDOTHELIAL NO AND PROSTAGLANDIN RELEASE

Fromy B., Merzeau S., Abraham P., Saumet J.L.

A significant transient increase in cutaneous laser Doppler flow during local external pressure application (11.1 Pa sec-1) was studied in the skin of rats, and defined as pressure-induced vasodilation (PIV). The aim of the present study was to examine the mechanisms involved in the efferent pathway of PIV, by testing whether the resultant vasodilation is endothelium dependent. The involvement of prostaglandins was tested in rats treated with indomethacin (5 mg kg-1, i.p.). Separate groups of adult rats were treated with either NG-nitro-L-arginine (20 mg kg-1, i.v.) or 7-nitroindazole (50 mg kg-1, i.p.) to inhibit nitric oxide synthase (NOS) activity and specific neuronal NOS, respectively. Prostaglandin inhibition resulted in a significant decrease in PIV (P<0.001 vs. vehicle). Inhibition of NOS abolished PIV (P<0.001 vs. vehicle), whereas specific inhibition of neuronal NOS showed diminution in PIV (P<0.001 vs. vehicle). These data suggest that PIV involves a contribution from prostaglandins and is dependent on endothelial NO, whereas neuronal release of nitric oxide has a smaller role.

Laboratory of Physiology, Medecine school, Rue Haute-de-Reculée, 49045 ANGERS - FRANCE

P03-18

AMBIENT TEMPERATURE AFFECTS THE CUTANEOUS PRESSURE-INDUCED VASODILATATION IN HUMANS

Koitka A., Saumet JL., Abraham P.

We have previously shown that during progressive moderate pressure strain a transient pressure-induced vasodilation (PIV) exists at the hand in normal subjects, but we failed to find a comparable response at the foot level. Previous works have found a significant sensitisation of vertebrate mechanoreceptors by temperature. The aim of our study was to examine the putative influence of the thermoregulatory state on skin blood flow responses to non noxious mechanical stimulus. We studied 10 healthy human subjects exposed to different ambient temperatures: cool (25.1±0.2°C), intermediate (27.0±0.4°C) and warm conditions (30.6±0.3°C). We measured skin blood flow using laser Doppler flowmetry on the head of the first metatarsus in response to a progressive local pressure increased of 5.0 mmHg/min. Progressive pressure strain led to an almost linear cutaneous laser Doppler flow (LDF) decrease in both cold and intermediate conditions, whereas in warm conditions subjects responded with a PIV. Indeed, mean resting LDF was 57.1±6.8 arbitrary units (a.u.), 75.2±7.7 a.u., 100.6±9.7 a.u at cool, intermediate and warm conditions respectively. In cold, intermediate and warm conditions, compared to baseline mean LDF at 30 mmHg was decreased to 33.4±6.1 a.u. and 50.9±6.2 a.u. and was increased to 134.3±16.7 a.u. (P<0.05 vs. baseline respectively). The data indicate that at PIV exists at the foot level in healthy subjects but the thermoregulatory state profoundly influences the extent and direction of vasomotor response to non-noxious pressure strain which is initiated by capsaicin-sensitive nerve terminals in the human skin. The results of these experiments suggest that the ambient temperature will affect discharge in the mechanical C-fibers involved in the PIV. Furthermore, there were no differences between responses in human hands and feet, suggesting an ubiquitary organisation of this original protective cutaneous reflex.

Laboratory of Physiology, Medecine school, Rue Haute-de-Reculée, 49045 ANGERS - FRANCE

P03-19

EARLY DECREASE OF SKIN BLOOD FLOW IN RESPONSE TO LOCALLY APPLIED PRESSURE IN DIABETIC SUBJECTS

Demiot C., Fromy B., Abraham P., Bouvet C., Bouhanick B., Fressinaud P., Saumet J.L.

Pressure ulcers are common debilitating complications of diabetes due to tissue ischemia. The skin blood flow in response to locally applied pressure might be impaired in diabetic patients due to combined effects of a typically low skin temperature and alterations of microcirculatory function, which could be worsen by neuropathy. We measured skin blood flow by laser Doppler flowmetry over the internal anklebone in response to a local pressure applied at 5.0 mmHg.min-1 in three groups of diabetic patients (with clinical, with sub-clinical and without neuropathy) and in healthy matched controls at usual room temperature. Compared to matched controls with comparable skin temperatures (29.3±0.4°C vs. 28.7±0.4°C), the skin blood flow response to locally applied pressure was further impeded in diabetics even without neuropathy. Indeed, skin blood flow decreased significantly from baseline at much lower applied pressure in diabetics, even without neuropathy (7.5 mmHg), than in controls (48.8 mmHg). The large difference between these pressures could partially explain the high risk of the occurrence of decubitus and plantar ulcers in diabetes.

Laboratory of Physiology, Medecine school, Rue Haute-de-Reculée, 49045 ANGERS - FRANCE

P03-20

EFFECTS OF NATURAL AND ENVIRONMENTAL ESTROGENS ON CELL SIGNALLING IN MUSSEL IMMUNOCYTES

Canesi L., Ciacci C., Lo Russo L.C., Betti M., Marchi B., Gallo G.

There is increasing evidence that immune cells represent preferential targets of the rapid, non genomic effects of both natural and environmental estrogenic compounds.

In bivalve molluscs, such as the mussel Mytilus galloprovincialis Lam., circulating hemocytes, that resemble the monocyte/macrophage lineage, are responsible for innate immunity. In this work the effects of the natural estrogen 17beta-estradiol on hemocyte cell signalling were evaluated, and the results were compared with those of synthetic estrogens (such as DESB), estrogenic chemicals (such as BisphenolA, PCBs) and plant estrogens (genistein). The results indicate that low nM concentrations of 17beta-estradiol induced a rapid, dose dependent increase in cytosolic [Ca2+] in Fura2/AM-loaded mussel hemocytes. Moreover, Western Blot analysis show that 17beta-estradiol affected the phosphorylation state of components of both the MAPK (Mitogen Activated Protein Kinase) and of the STAT (Signal transducers and Activators of Transcription) family, whose activation has been demonstrated to play a crucial role in mediating the transduction of bacterial signals in mussel hemocytes. Higher concentrations of 17beta-estradiol were toxic to hemocytes, resulting in significant destabilisation of lysosomal membranes. Both synthetic and environmental estrogens mimicked the effects of 17beta-estradiol; however, similar effects were observed at concentrations 1000 times higher (microM) than those of the natural estrogen. Overall, our data demonstrate that in invertebrate cells both natural and environmental estrogens can act through rapid, non genomic mechanisms affecting both Ca2+- and tyrosine kinase-mediated cell signalling involved in mediating the innate defence response to bacterial stimuli.

DI.BI.S.A.A. Università di Genova, Genova , Italy

P03-21

CANDIDA ALBICANS PROMOTES ICAM-1 AND E-SELECTIN EXPRESSION THROUGH A NFKB MECHANISM

Claveau I., Mostefaoui Y., Rouabhia. M.

Candida species are the most frequent cause of life-threatening invasive fungal infections in the immunocompromised host and are responsible for 10% of all nosocomial bloodstream infections. The leading cause of candidiasis is Candida albicans. Adhesion to the epithelium is the first step toward colonisation of the oral mucosa by this fungus. Oral epithelial cells hence play a crtitical role in maintaining the equilibrium between host cells and C. albicans. The aim of this study was therefore to investigate the early events following contact between epithelial cells and C. albicans. To this end, the expression of ICAM-1 and E-selectin by oral epithelial cells and the signal transduction pathways following C. albicans infection have been studied. In this context, an engineered human oral mucosa has been produced, then infected with C. albicans ( 10ex5 yeast/cm2). At the end of each appropriate contact period, total mRNA and proteins were extracted from oral epithelium and then used for RT-PCR, immunohistochemistry and Western blot analyses. Our results demonstrate that C. albicans significantly up-regulates the mRNA expression of ICAM-1 and E-selectin after 24 h of infection. ICAM-1 and E-Selectin were promoted by mitogen-activated protein (MAP) kinase cascade (ERK1/2, JNK1/2, p38, cJUN, ATF-2). Indeed, C. albicans modulates the phosphorylation pattern of these MAP kinase proteins and the expression of EGFR and NFkB. In conclusion, this study made some light on the mechanism involved in C. albicans adhesion to oral epithelial cells. (Funded by the FRSQ, NSERC and CIHR)

Faculté de médecine dentaire/GREB, Université Laval, Quebec City, Quebec, Canada G1K

P03-22

Hg2+-DEPENDENT CELL SIGNALLING IN TROUT HEPATOMA (RTH-149) CELLS

Viarengo A., Burlando B., Magnelli V., Bonomo M., Berti E.

The study of ligand-independent cell signalling is a promising field for an understanding of cellular responses to stress. We report here different signalling events triggered by Hg2+, a strong reagent of sulfhydryl and imidazole groups, on the RTH-149 trout hepatoma cell line.

Confocal imaging of fluo 3-loaded cells showed that Hg2+ triggered [Ca2+]i transients and Ca2+ waves. The [Ca2+]i rise was reduced by the Ca2+ channel blocker verapamil and abolished by extracellular glutathione (GSH), but it was almost unaffected by cell loading with the heavy metal chelator TPEN or with esterified GSH. In Ca2+-free medium, Hg2+ induced a lower [Ca2+]i transient, that was abolished by manoalide, a PLC inhibitor, or by cell loading with GDP-S or heparin. Also, cells loaded with heparin and exposed to Hg2+ in the presence of external Ca2+ showed a drastic reduction of [Ca2+]i rise. Data indicate that Hg2+ induces both extracellular Ca2+ entry and InsP3-dependent intracellular Ca2+ release. These two processes are not independent, as Ca2+ release is amplified by Ca2+ entry through Ca2+-induced Ca2+ release.

Western immunoblotting of cell lysates and the use of antiphosphotyrosine showed that Hg2+ induced an increase of tyrosine phosphorylation. Pre-incubation with genistein did not abolish this effect but only reduced it, probably due to tyrosine kinase stimulation coupled to phosphatase inhibition. The use of phosphospecific antibodies against MAPKs, representing stress-activated tyrosine kinase signalling pathways, showed strong activation of ERK and p38, and a lower activation of JNK.

Our results indicate that short-term effects of Hg2+ consist in the activation of both calcium and phosphotyrosine signalling, possibly due to Hg2+ interactions with plasma membrane receptors. Hg2+-dependent signalling could play a role in the activation of defense mechanisms able to protect cells after metal upload.

Dipartimento di Scienze e Tecnologie Avanzate, Università del Piemonte Orientale ,15100 Alessandria, Italy

P03-23

EFFECT OF ANTIOXIDANT ADMINISTRATION ON LIVER FUNCTION AFTER COLD PRESERVATION

Ben Abdennebi H., Saïdane D. ., Mrabet I., Steghens J.P., Virieux S., Gharib C.

One of the main causes of the grafts loss after liver transplantation is due to the ischemia-reperfusion injuries. In that case, it is clinically important to elucidate the mechanism of cellular damage during hepatic preservation and reperfusion and to improve a new preservation solution and a rinse solution. Our aim was therefore to evaluate the usefulness of enriched solutions for rinsing liver after cold preservation.

The isolated perfused rat liver (IPRL) model was used to assess organ recovery. After 24h of cold preservation in university of Wisconsin (UW) liquid and 30 min of warm ischemia, livers were perfused for 2h at 19 °C with Krebs-Henseleit solution (KH, n=7) or KH+antioxidants (n=6) or KH+nifedipine (n=6). The results showed that antioxidants addition to KH induced an increase of bromosulfoptalein (BSP) clearance (p<0.05 vs KH and KH+nifedipine) and a decrease of aspartate aminotransferase (p<0.05 vs KH and KH+nifédipine) and alanine aminotransferase (p<0.05 vs KH) release into perfusate. In addition, intrahepatic resistances are improved (p<0.05 vs KH and KH+nifedipine) with antioxidants.

In conclusion, antioxidants given during the early post-preservation period improve liver graft function. During cold storage, energy depletion leads to an impairment of cellular homeostasis and several toxic mediators are released. The use of an enriched solution with antioxidants to rinse grafts after cold storage allows loading the organ with protective compounds, which could be essential for recovery after reperfusion.

* This study was supported by grant (MIRA 2001) from le Conseil Régional Rhône-Alpes - France.

Service de Physiologie Humaine, Faculté de Pharmacie, 5000 Monastir, Tunisie

P03-24

EXERCISE PERFORMANCE IN SUBJECTS WITH SICKLE CELL TRAIT IN HOT CLIMAT

Samb A., Gueye L., Seck D., Cissé F., Badji L., Martineaud J P.

The sickle cell trait is a genetic abnormality of the red blood cell. It is due to the mutation of a parental gene, one amino-acid of the chain ß of the globin a glutamic acid which is substituted by a valin on the haemoglobin (HbAS). For subjects with sickle cell trait (SCT). The possibility to display any disturbance during predominantly anaerobic and aerobic exercises is unclear.

19 subjects with sickle cell trait and 19 subjects control have been studied during incremental exercise test on cycloergometer and on aera. They are all students of the Institut National Supérieur d’Education Populaire et Sportive of DAKAR. The environmental temperature mean has been 26°C. After haematological analysis an incremental exercise has been performed during 15 or 20 mn for one group. For another group a sub maximal muscular exercise for one hour with 75% of maximal heart rate has been done. We have determined VO2max , heart rate, blood pressure, rectal and skin temperature during exercise.

Haematological analysis has shown any different between the two groups. Any difference was found in VO2max and cardio circulatory variables during maximal exercise in cycloergometer between control group and sickle cell trait group VO2max was at mean 44.7±8.1 vs 44.6±6.9 ml/mn/kg respectively. The two groups have done sub maximal exercise during 1 hour without difficulty. We have not observed any difference between the two groups in cardiovascular, thermal variables and developed mechanic power. Rectal temperature for control group and SCT group was at mean 37.86±0.23 vs 37.93±0.27°C respectively and 33.92±1.91 vs 32.5±2.1°C for skin temperature

These results show that subjects with SCT have exercise performance comparable with control subjects during incremental maximal exercise and sub maximal exercise for 1 hour. We can assure that subjects with SCT in our country may participate in sports competition, as well as subjects with normal HbAA

Laboratoire de physiologie Faculté de Médecine Dakar (SENEGAL)

P03-25

THE QUALITATIVE CHANGES OF RED BLOOD IN HYPOXIA

Sumin M., Rezaikin A., Salimov D., Yushkov B.

The quantitative and qualitative changes of red blood in hypoxia were complexly studied.

The objects of research were white rats. The hypoxia was equivalent to seven thousand meters above the sea level. It was achieved by rats' incubation in the barochambers during seven days (6 hours daily). We estimated the level of hemoglobin, concentration of erythrocytes, reticulocytes in the blood and erythroid cells in the bone marrow. The size of erythrocytes was measured. Two methods was used to determine the hemoglobin's pattern: electrophoresis in polyacrylamid gel and acid elution method.

After the seven day of hypoxia the blood level of five and six hemoglobin's fraction (these are fetal isoforms of rat's hemoglobin) was increased. It was accompanied with the large cells appearing in the blood circulation. The measurement of erythrocytes with acid stable fractions of hemoglobin (five and six isoforms) showed that these were the largest. It is known that the size of erythroid cells is decreasing during it maturation. So it can be supposed that the higher level of five and six hemoglobin's fractions characterize one of the erythroid cell maturation stage. However the hemoglobin's pattern of extracted reticulocytes didn't differ from the one of other age cells. Besides we ascertained that old rats with depressed erythropoiesis had the high level of five and six hemoglobin's isoforms in the blood circulation.

Therefore the macrocytes with the higher level of five and six fractions of hemoglobin is not the stage of erythroid maturation. Probably there are some ways of red blood's formation. The first way is inherented to adult healthy animal. Another way prevails in fetal and neonatal period, in old age and in hypoxia. So hypoxia leads to qualitative erythropoiesis' changes consisted in production of macrocytes with higher level of five and six hemoglobin's isoforms (fetal isoforms).

Immunology and Physiology's Institute of Ural Branch of Science's Russian Academy -RUSSIA

P03-26

EFFECTS OF COLD-RESTRAINT STRESS-INDUCED LIPID PEROXIDATION ON RAT PERITONEAL MACROPHAGES FUNCTIONS

Tan R., Bulbul M., Izgut-Uysal V.N.

The aim of this research was to study the effects of cold restraint stress-induced

lipid peroxidation on the phagocytic and chemotactic capacities of peritoneal macrophages from rats. Macrophages were obtained with peritoneal lavage from control and stress groups. The rats in stress group were exposed to cold and restraint stress for 4 hours at +4ºC. TBARS formation and catalase activity were measured in peritoneal macrophage suspensions. Phagocytosis of macrophages was evaluated according to the mean number of phagocyted particles, and chemotaxis in a Boyden chamber. In stress group, catalase activity and TBARS production were higher than the control animals but this difference was not significant. Chemotactic and phagocytic capacities of macrophages reduced significantly in the stress group. In conclusion cold-restraint stress decreased the phagocytic and chemotactic activities of peritoneal macrophages from rats.

Department of Physiology, Faculty of Medicine, Akdeniz University, Antalya, Turkey

P03-27

EFFECT OF THYROID HORMONE IN A STUDY ON ADULT AND FETAL SYNGENEIC HEPATOCYTE TRANSPLANTATION

Cubero F.J. (1), Mula N. (1), Codesal J. (2), Cantarino M.H. (3), Garcia-Barrutia M.S. (3), Maganto P. (1), Arahuetes R.M. (3)

AIM:The aim of this work is to assess the survival and functionality of syngeneic adult and fetal hepatocytes transplanted into Gunn rats, an experimental model of the syndrome of Crigler-Najjar type I, and to evaluate the ability of thyroid hormone (T3) to stimulate liver repopulation.METHODS:Hepatocytes were isolated by collagenase digestion and transplanted into spleens of Gunn rats. These were divided into four experimental groups: I) Animals receiving adult hepatocyte transplantation (THC) without T3. II) Animals receiving fetal hepatocyte transplantation (THF) without T3. III) Animals receiving THC and the same day and every 10 days thereafter, T3 (Sigma; 400 mg/100 g body weight) injected subcutaneously. IV) Animals receiving THF and treatment with T3. Controls consisted of untreated rats and treated with T3 but without transplantation. Rats were killed from 1 to 15 days after implants and livers and spleens were processed by histological methods. Also, bilirubin levels were assessed from blood and bile at the end of the study. RESULTS:Although all groups show a significant decrease in serum total bilirubin, the percentage of decrease in animals transplanted with either adult or fetal hepatocytes and treated with T3 is significantly greater. In both THF and THC stimulated with T3, there is a dramatic decrease of total serum bilirubin by day 15 after the implant which is coincident with an increase of conjugated bilirubin in bile. Light microscopy study shows that transplanted cells migrate to the liver of recipient animals.

CONCLUSION:In short, it seems that repeated injections of T3 might provide a strong stimulus for transplanted cells to repopulate the liver as indicated by the decrease in bilirubin being the effect most remarkable 15 days after two injections of thyroid hormone. The use of proliferating agents could be an alternative to repeated implants for correcting a metabolic diseases. (This work was financed by grants FIS 01/0001-01 and 02)

(1)Clinica Puerta de Hierro - (2)Universidad Autónoma - (3)Universidad Complutense. Madrid. SPAIN

P03-28

PLASMA REPRODUCTIVE STEROID AND MORPHOLOGY OF THE GONAD IN THE FLOUNDER (PLATICHTHYS FLESUS)

Durán B., Damasceno-Oliveira A., Coimbra J.

The objective of this study was to establish the different stages of the gonadal development and the associated steroid hormone levels in adult males and females of a commercially important flatfish species, Platichthys flesus, during its annual reproductive cycle and its migrations from feeding grounds to spawning grounds, from estuarine waters (fresh and brackish water) to coastal waters (sea water).

The animals were captured in the estuary of the river Douro (Porto-Portugal) and adjacent coastal waters during a period of time of seven months, starting from Setember until November. The sampling times were chosen to coincide with the period of gonadal recrudescence and spawning. Blood was collected from the caudal vein for sexual steroid analysis with a radioimmunoassay (RIA), 17b-estradiol in females and testosterone in males. Gonadal tissue samples were collected and taken for histological examination. These were fixed with Smith´s fixative, embedded in paraffin, stained with Gill hematoxylin and eosin and examined by light microscopy.

The results obtained in this study allow to describe and to interpret the different phases of the gonadal development that take place in different enviromental conditions. It was observed that this species has an annual reproductive cycle, beginning in autumn and with spawning in spring. The variations in plasma reproductive steroid concentrations are correlated with the morphologic changes of the gonad.

Acknowledgements.- This work was partially funded by Fundação para a Ciência e Tecnologia, Portugal, through the project "Flatfish spawning migrations : phisiologycal aspects (POCTI/BSE/41025/2001)».

Centre of Marine and Environmental Research (Ciimar), University of Porto, Portugal

P03-29

EFFECT OF MAXIMAL MUSCULAR EXERCISE ON TNF-α : ATHLETE – SEDENTARY COMPARISON

Tabka Z., Denguezli M., Debbabi H. ., Ben Jabrallah M., Gaied S., Chouchane L.

Tumour necrosis factor-alpha (TNF-α) is a multi-functional cytokine that arouses a particular interest at the fundamental level as well as at the clinical one. Initially described for its anti tumour property, this agent reveals it self as an important mediator of inflammation, besides, it has been shown that it is associated to many different auto-immune infectious and tumoral diseases.

The aim of this study is to identify the effect of maximal muscular exercise on the plasma concentration of TNF-α.

The experiences concerned healthy volunteers : seven athletes and eight sedentary, each of them underwent a triangular effort exercise on an ergometer bicycle.

Two blood samples were taken, one before, and the other immediately after the exercise. The plasma concentrations of TNF-α were measured using an enzyme-linked immunosorbent assay (ELISA).

This measurement showed a significant increase of TNF-α plasma concentration both for the athlete and sedentary subjects (before 20.37 pg/ml ± 25.39 ; after 123.57 pg/ml ± 120.35) (before 18.33 pg/ml ± 19.74 ; after 54.59 pg/ml ± 67.92). This increase is much more significant as for athletes. It is estimated at 500% versus 200% for the sedentary.

During an intense physical activity, the muscle is mechanically injured and this process stimulates the liberation of inflammatory cytokines by tissular macrophages.

A continual production of TNF-α engenders an excessive inflammatory response followed by an increase of the prostaglandin and of the stress hormones secretion, modifying, by the same way, the cytokinic responses profile and this results in a less efficient immunitary responses, a process which increases upper respiratory tracts infect a few hours or days after the exercise.

Faculté de Médecine IBN EL JAZZAR, Sousse, Tunisia

P03-30

ION TRANSPORT AND BIOMINERALIZATION IN SCLERACTINIAN CORALS

Allemand D., Bouchot A., Puverel S., Tambutté E., Tambutté S., Zoccola D.

Whereas scleractinian corals are one of the major calcifying groups of organisms in the living world, calcification processes largely remain a biological enigma. Within the organic matrix, calcification consists of the precipitation of CaCO3 according to the equation Ca2+ + HCO3- -> CaCO3 + H+.

Calcification occurs in a "biologically-controlled medium" at the innermost margin of the ectodermal (calicoblastic) cells of the aboral layers, consequently, to reach the skeleton, ions have to cross oral and then aboral cell layers.

Experiments were conducted on Stylophora pistillata microcolonies.

We demonstrate that Ca2+ ions cross the oral layers by a diffusive, paracellular pathway. Experiments with channel inhibitors show that Ca2+ transepithelial transport involves at least one transcellular pathway across the calicoblastic cells. This pathway involves L-type Ca2+ channel proteins which alpha1 subunit has been cloned and immuno-localized on the calicoblastic cells. Identities and conservative substitutions between the rabbit alpha1C-subunit and the coral Ca2+ channel are 52.5% and 86% respectively, demonstrating the conservation of ionic supports through evolution. Concerning Ca2+ export from the calicoblastic cells to the skeleton, we have cloned a Ca-ATPase gene. Phylogenetic reconstruction shows that the pump is closely related to the PMCA family found in vertebrates. By fluorescence in situ hybridization we show the preferential expression of the pump within the aboral tissue.

Regulation of biomineralization also implies the fine control of pH. Using an antibody raised against a P-type proton-ATPase of yeast, we observe a specific staining of the calicoblastic epithelium. We hypothesize that this protein is necessary for the elimination of H+ resulting from the deprotonation of HCO3- during CaCO3 precipitation. We actually purify this carrier.

We thus propose a model for ion transport and outline the role of calicoblastic cells in coral calcification.

Centre Scientifique de Monaco - Monaco



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