VOLTAGE GATED ION CHANNELS IN TRANSDUCTION AND ADAPTATION IN CRAYFISH STRETCH RECEPTOR.
Rydqvist B., Swerup.C., Sand P.
The crayfish stretch receptor, an analogue to the human muscle spindle, is a classical model for the study of transduction in a mechanoreceptor. Analysis has shown that viscoelastic properties and mechano-gated channels are important determinants of transduction. However, voltage gated ion channels permeable to Na+ and K+ also contribute to the overall response of these sensory receptors and in particular to explain the difference in adaptive behaviour of the slowly and rapidly adapting neurone. To further investigate the transduction of these receptors we have studied the different ion channels present in these neurones and the possible spatial distribution of the voltage gated ion channels. Two electrode voltage clamp and patch clamp experiments in the stretch receptor neurones of the crayfish (Pacifastacus leniusculus) have demonstrated that the Na+ channels are differently distributed in the two neurones. In the slowly adapting neurone the Na+ channels seems to be present in both axon and soma whereas in the rapidly adapting neurone the Na+ channels are present in the axon only. Three different types of K+ channels are present in the neurones. One of them have been characterized as an outward rectifying channel of type Kv1.2. Two other potassium channels are of the transient type. Mathematical modelling also confirms that small changes in activation inactivation properties of the Na+ and K+ channels and spatial distribution result in considerable difference in adaptive properties.
It is concluded that the main voltage gated ion channels have a decisive importance for the adaptive properties of these neurones when stimulated both mechanically and electrically.
Dept Physiology & Pharmacology, Karolinska Institutet, S-17771 Stockholm, Sweden
PROTEIN KINASE A AND PROTEINE KINASE C MODULATION OF NAV1.7 AND NAV1.8 NERVE SODIUM CHANNELS.
Vijayaragavan K., Chahine M.
Voltage-gated sodium channels (VGSC) are transmembrane proteins essential for initiation and propagation of action potentials in neuronal excitability.
Dorsal root ganglion specific VGSC Nav1.7 and Nav1.8, were expressed in Xenopus oocytes and the effects of protein kinase activation on
the Na+ currents were studied using the two-electrode voltage clamp method. Our data show that PKC and PKA differentially regulate these channels.
A dose-dependent attenuation of Na+ current is observed when phorbol esters are applied to both channels, Nav1.8 being more sensitive.
In addition, a 6mV shift of only the voltage-dependence of activation towards more depolarized potentials is observed for Nav1.7.
However, no shift of steady-state gating is observed for Nav1.8. The Nav1.8 decrease in peak current can be inhibited with epsilon PKC antagonist, while epsilon PKC and betaII PKC both seen to modulate the PMA induced effect on Nav1.7. PKA activation instead results in a dose-dependent increase in Nav1.8 Na+ current
but decrease in Nav1.7 Na+ current with no shifts in voltage-dependence of gating.
The PKA-mediated rise of Nav1.8 Na+ current is inhibited by chloroquin that affects vesicular trafficking. This suggests that during nerve injury, increased PKA activity could enhance Nav1.8 trafficking to the synaptic membrane surface, which may cause C-fiber hyperexcitability.
However the functional consequence of these channels would depend on the fine balance between the activated PKA and PKC isozymes.
Department of medicine, Laval University, and Laval hospital, Research Centre, Sainte-Foy, Canada
L 703,606 AS MODULATOR OF ELECTROGENIC IONIC TRANSPORT IN COLON
Mlodzik N., Lelinska A., Kaczorowski P., Tyrakowski T.
The C-fiber endings in the colon influence local physiological functions of the intestine by releasing of sensory peptides such as substance P, NKA and NKB.
In this study the effects of L 703,606 (the NK-1 antagonist) transepithelial potential difference were examined.
The experimental model was an isolated colon wall mounted in Ussing apparatus. The mechanical stimulation of C-fiber endings was by gentle rinsing of mucosal surface of the colon by jet-flux from peristaltic pump. The 35 specimens of isolated colonic walls from 11 rabbits were investigated. Every significant reaction was repeated at least ten times.
After mechanical stimulation the hyperpolarization of the tissue was noticed. In the presence of chloride transport inhibitor - bumetanide L 703,606 in the concentration of 10-7 M and 10-6 M were able to augment electrogenic ion current and in the concentration of 10–5 M were able to diminished the electrogenic ion current.
These electrophysiological data evidenced that colonic receptors for tachykinins influence electrogenic ion transport differently: augmenting it in smaller concentration and diminishing it in higher concentration. The hypothetical mechanism for opposite effects of the different concentrations of drug is the presence of the two different populations of tachykinin receptors in colon - autoreceptors on C-fiber ending (responsible for the augmentation of the reaction) and epithelial receptors (responsible for its diminution).
Patobiochemistry and Clinical Chemistry, L. Rydygier Medical University, Bydgoszcz, Poland
A NEW EXPRESSION SYSTEM FOR PANCREATIC Na+/Ca2+ EXCHANGER NCX1.3 AND NCX1.7
Hansen MR., Amstrup J., Novak I.
Activity of the Na+/Ca2+ exchanger (NCX) in rat pancreatic ducts is regulated by pancreatic secretagogues secretin, acetylcholine, insulin and ATP. The studies were carried out on intact rat pancreatic ducts, which express the NCX splice variants NCX1.3 and NCX1.7. Therefore regulation of Na+/Ca2+ exchange can be due to any of these variants. In order to separately study NCX1.3 and NCX1.7, we require a model system with cell lines expressing those individually. The aim was therefore to generate plasmid constructs with rat pancreatic NCX1.3 and NCX1.7 cDNA.
Using an RT-PCR based approach on rat heart and pancreatic RNA, six different constructs with hybrid NCX1.3 and NCX1.7 cDNA coupled to Enhanced Green/Blue Fluorescent Protein (NCX1.3-EGFP, EGFP-NCX1.3, EBFP-NCX1.3, NCX1.7-EGFP, EGFP-NCX1.7 and EBFP-NCX1.7) were generated. The differential tagging allows us to assess the effect of tagging the N- or C-terminal part of the protein and to visualize co-localization of NCX1.3 and NCX1.7 chimeras in transfected cells. The constructs were successfully expressed in HEK293 cells. Expression of all six constructs and targeting of the NCX1.3-GFP chimera to the plasma membrane was verified with western blotting and confocal laser scanning microscopy, respectively. RT-PCR was used to check for expression of NCX isoforms, purinergic receptors and Ca2+ -binding proteins in model cell lines HEK293 and Capan-1 to compare the background of the cell lines with the known expression in native pancreas. Using the above mentioned constructs expressed in HEK293 and Capan-1 we are now carrying out experiments using fluorescent optical techniques to study the function and regulation of the pancreatic Na+/Ca2+ exchangers. Thus, a new expression system useful for physiological studies on the function and regulation of pancreatic Na+/Ca2+ exchange has been established.
August Krogh Institute, University of Copenhagen, Copenhagen, Denmark
P2X RECEPTOR MEDIATED INTRACELLULAR SIGNALLING
Amstrup J., Novak I.
Nucleotides are extracellular agonists of specific purinergic P2 receptor subtypes. The P2 receptors are well characterized by pharmacological and electrophysiological methods. However, relatively little is known about their intracellular signalling pathways, especially those via P2X type receptors that are widely expressed in exocrine glands. The coexistence of different types of P2 receptors, together with a general lack of selective agonists and antagonists, has made advances in P2 receptor characterization and P2 mediated signal transduction difficult using native tissues and physiological methods. The aim of the present study was to set up a model system using heterologous expression of the P2X4 receptor in HEK293 cells and study the signal transduction pathway(s).
Inserting cDNA coding for the P2X4 receptor into vectors containing a green fluorescent protein (GFP) at either the N- or C-termini of the receptor enabled us to detect the receptors by Western blot and by use of confocal laser scanning microscopy (CLSM).
CLSM studies showed that the GFP-P2X4 chimera was targeted to the plasma membranes in HEK293 cells. Stimulation of transfected HEK293 cells with 30 M ATP mediated an activation of p44/42 MAP kinases. Further, we investigated which parts of the P2X4 receptor were involved in this activation by making constructs lacking part of either the intracellular C- or N-terminals. The constructs lacking a part of the intracellular N-terminal were still able to mediate an ATP activation of the p44/42 MAP kinases. In contrast, the construct missing the C-terminal part of the P2X4 receptor, were not able to mediate activation of p44/42 MAP kinases. Furthermore, an ATP-mediated tyrosine phosphorylation of the P2X4 receptor was detected. In conclusion, these experiments suggest that the C-terminal part of the P2X4 receptor is involved in ATP-induced activation of p44/42 MAP kinases, and that the receptor is tyrosine phosphorylated in response to ATP.
August Krogh Institute, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen, Denmark
STIMULATION OF Na-K-2Cl CO-TRANSPORTER BY MOLECULAR INTERACTION WITH AE1 IN XENOPUS OOCYTE.
Guizouarn H., Gabillat N., Borgese F.
Regulation of membrane permeability could be achieved by direct modifications on transporters (as phosphorylations) or by interactions between transporters. These interactions could be functional (electric coupling, thermodynamic coupling or autocrine mechanism...) or they could also involve direct contacts between proteins.
An example of membrane transporters regulation by protein interactions that are not related to transport functions is given by the trout anion exchanger, tAE1, and the Na-K-2Cl co-transporter.
Xenopus oocyte expressing tAE1 for one day exhibits a strong Na and Cl-dependent Rb influx that is mediated by the endogenous Na-K-2Cl co-transporter. Other members of the AE1 family (skate or mouse AE1) were not able to stimulate the co-transporter. Our data previously showed that tAE1 but not other AE1, induced a Cl channel in oocyte. It was possible to specifically inhibit with glybenclamide the tAE1 anion channel without affecting stimulation of the Na-K-2Cl co-transporter. Measurements of intracellular Na+, K+ and Cl- concentrations in control or tAE1 expressing oocytes could ruled out involvement of ion content modifications in co-transporter activation. Moreover, activation of the Na-K-2Cl co-transporter by tAE1 expression was abolished by alteration of the C-terminal end of tAE1. These alterations were obtained either by reaction with a specific antibody raised against the last amino-acids of tAE1 or by fusion of gyrase B to the carboxy terminal end of tAE1. By the use of chimeric AE1, it was possible to conclude that tAE1 stimulation of the co-transporter is not linked to tAE1 conductive properties but rather to interaction between the C-terminal end of tAE1 and the Na-K-2Cl co-transporter.
This interaction observed in tAE1 expressing oocytes could take place in physiological conditions to coordinate activity of different transporters and regulate trout erythrocyte membrane permeability.
LPMC. UMR 6078/CNRS-UNSA. 284 chemin du Lazaret, 6230 Villefranche/Mer, France
INHIBITION OF THE MUCIN PRODUCTION BY ANION AND V-ATP-ASES BLOCKERS IN THE NCI-H392 CELL LINE
Chénafi O., Bogliolo S., Renard C., Bernard K., Ehrenfeld J.
Mucus overproduction is an important feature of airway diseases including cystic fibrosis, chronic obstructive pulmonary disease or asthma. Recently, a calcium-activated chloride channel, hCLCA1, was proposed to be responsible, in part, for the overproduction of mucus in asthmatic subjects. These preliminary findings suggest the inhibition of hCLCA1 may be an important new therapeutic approach to control mucus overproduction in chronic airway disorders. Up to now, the functional role of Cl- channels in this process is unknown. We postulated that acidification of secretory mucin granules implicates the function of Cl- channels associated with V-type H+-ATPase and used NCI-H392, a cell line derived from a human pulmonary mucoepidermoid carcinoma, as a model of mucin secretion. An immunoassay of the MUC5AC protein was used to evaluate the effects of known anion channel and proton V-ATPase inhibitors on the mucin production in these cells. The involvement of K channels also present in secretory granules was also investigated.
Epidermal grow factor (EGF) application induced a three fold increase in the mucin synthesis and in the mucin secretion. Simultaneous application of EGF with one of classical anion channel inhibitors (NPPB, NFA, DIDS, DPC or glybenclamide) resulted in an inhibition of mucin synthesis without significant effect on mucin secretion. Charybdotoxin and clotrimazole, two KCa (SK4) channel inhibitors or chromanol 293B, a KcAMP channel blocker were ineffective on mucin production. The H+ pump inhibitors, DCCD and oligomycin as the more specific V-ATPase blocker bafilomycin considerably reduced the EGF-stimulated mucin synthesis and totally abolished the mucin secretion. hCLCA2 as hCLCA4 mRNAs were detected by RT-PCR but not hCLCA1 transcripts. We conclude to a key role of central vacuolar V-ATPase in the process of mucin synthesis and secretion. Cl- channels participate to the first event but their molecular identification remains to be precised.
UMR 6078 (UNSA/CNRS) LPMC, Villefranche sur mer, France
PARACELLULAR REGULATION OF ION ABSORPTION IN NATURAL AIRWAY EPITHELIUM
Frederiksen O., Poulsen A.N., Willumsen N.J., Pedersen P.S.
Fast regulation of the depth of upper airway apical surface fluid layer (ASL) is accomplished by regulation of NaCl absorption in the airway surface epithelium. Natural airway epithelium is leaky and Na+ absorption through ENaC channels is accompanied by passive Cl- absorption through an anion selective paracellular pathway. In the present study we investigated the relative importance of regulation of cellular and paracellular pathways for the downregulation of NaCl absorption by luminal ATP/UTP.
Short circuit current (ISC), epithelial conductance (Gt), and tracer fluxes of Na+, Cl-, and mannitol were measured under short circuit conditions in native airway epithelium from the rabbit nasal septum mounted in Ussing chambers.
Mucosal nucleotides inhibited amiloride-sensitive Na+ absorption but only slightly increased Cl- secretion. From changes in Gt it was calculated that ATP/UTP caused a large decrease in paracellular conductance (GS) and in parallel passive (paracellular) Cl- fluxes (but not passive Na+ and mannitol fluxes) decreased. The effects of nucleotides were mimicked by ionomycin and pretreatment with ionomycin largely prevented the effects of ATP/UTP on ISC and Gt. Stimulation of cAMP by P1 (adenosine) receptor stimulation or by forskolin only slightly affected ISC but increased Gt to an extent that involved a substantial increase in GS.
The results suggest that ATP and UTP released to ASL exert an autocrine regulatory function on native airway epithelial ion transport, primarily by inhibiting net NaCl absorption, while stimulation of Cl- secretion is of minor importance. This downregulation is caused by an activation of apical P2Y2 receptors leading to an increase in [Ca2+]i which inhibits apical ENaC channels and paracellular anion (Cl-) permeability. The permeability of the anion-selective paracellular pathway is under dual and opposite control from [Ca2+]i and cAMP.
Department of Medical Physiology, The Panum Institute, Copenhagen, Denmark
PANCREAS SECRETES PARTICULATE NUCLEOTIDASE CD39 – NEW ASPECTS OF PURINERGIC SIGNALLING
Novak I., Sørensen C.E., Amstrup J., Rasmussen H.N., Ankorina-Stark I., Møbjerg N.
Pancreatic acini release ATP and the excurrent ducts express several types of functional purinergic P2 receptors. Thereby, ATP might play a role as a paracrine regulator between acini and ducts. The aim of the present study was to elucidate whether this acinar-ductal signaling is regulated by nucleotidase/s, characterize and localize it within the rat pancreas.
In our studies we used physiological, biochemical and molecular biological methods to characterize nucleotidase activity in the rat pancreatic tissue and in pancreatic juice. RT-PCR and Western blotting revealed that the pancreas expresses the full length 78 kDa ecto-nucleoside triphosphate diphosphohydrolase, CD39. Immunofluorescence shows CD39 localization on basolateral membranes of acini, luminal membranes of small intercalated/interlobular ducts and basolateral membranes of larger ducts. Upon stimulation with CCK-8, acinar CD39 relocalized towards the luminal pole. Accordingly, pancreatic juice collected from intact pancreas stimulated with CCK-8 did not contain significant amounts of ATP, but nucleotidase activity, including that of CD39. Anti-CD39 antibodies detected a full length CD39 in pancreatic juice. This CD39 was only confined to the particulate and not the soluble fraction of the CCK-8 stimulated secretion. No CD39 activity was detected in the secretin-stimulated secretion. Electron microscopy shows that pancreas secretes microsomes that presumably contain the CD39 activity. The role of secreted CD39 would be to regulate intraluminal ATP concentrations within the ductal tree. The final product of ATP hydrolysis by CD39 and other nucleotidases is adenosine, and as we show by patch-clamp studies larger ducts possess adenosine receptors that regulate Cl channels. In conclusion, we show a novel inducible release of full length particulate CD39, and propose its role in physiological context of pancreatic secretion.
August Krogh Institute, University of Copenhagen, Denmark
CHLORIDE EFFECTS ON THE FUNCTION OF THE GABA COTRANSPORTER RGAT1
Pisani R., Giovannardi S., Fesce R., Bossi E., Binda F., Peres A.
The effects of reducing external Cl- on the electrophysiological properties of the Na+/Cl--dependent GABA transporter rGAT1 expressed in Xenopus oocytes were investigated. In agreement with a recently proposed kinetic scheme, the effects of Cl- are complex but preserve the mutual relationship that links the transport-associated currents, Itr, measured in saturating GABA concentration, and the transient current Ipre, recorded in the absence of GABA following a voltage step from the holding potential Vh to V. In particular Itr(V)-Itr(Vh)= r[integral]Ipre(V)dt, where r is the relaxation rate of Ipre at the same membrane potential and Cl- concentration. The model also predicts a relation between charge relaxation rate and apparent affinity for GABA, which is also verified in presence of lowered Na+ or Cl- concentrations. In these conditions the binding rate of GABA to the transporter is increased. All these effects are consistent with the hypothesis that interaction of the organic substrate with rGAT1 induces a conversion from a capacitive to a conductive mode of operation without strongly altering either the amount or the rate of charge movement.
Laboratory of cellular and molecular Physiology, DBSF, University of Insubria, Varese, Italy
NIFEDIPINE-ACTIVATED CATIONIC PERMEABILITY IN MDCK CELLS.
Melendez E. (*), Bidet M., Tauc M., Reyes J.L. (*), Poujeol P.
We have demonstrated that newborn rat distal cells express an apical Ca2+ channel that presents the characteristic to be activated by dihydropyridine drugs. With a similar approach (Fura2), we found that, in MDCK cells, nifedipine increases Ca2+i in a dose-dependent manner (IC50 = 4µM). The requirement of extracellular calcium was clearly established since this increase was abolished in EGTA containing solution. The Ca2+ channel antagonist isradipine as well as the agonist BayK8644 caused such an increase of Ca2+i indicating that this effect is related to the dihydropyridines as a substance class. Diltiazem (20µM) significantly inhibited the nifedipine effect (62% inhibition in calcium variation). Gadolinium (200µM) also had a significant inhibiting effect (43%). La3+ even at high concentrations (100 µM) was ineffective. Clamping membrane potential with valinomycin did not modify the nifedipine-induced Ca2+i increase, indicating that it was not related to potassium flux. Results obtained with fura2-loaded cells suggested that nifedipine activates an electrogenic mechanism. On that account, we performed whole cell clamp experiments. When MDCK cells were maintained at –50mV in a perfusion solution containing10 mM CaCl2, the addition of 20µM nifedipine induced an increase of the current (1.2±0.3nA) which, was inhibited by Gd3+. No significative current was observed when nifedipine was added in the presence of 0.5 mM EGTA. To precise the effects of nifedipine on the membrane potential, we then performed oxonol fluorescence experiments. Addition of nifedipine or BayK8644 induced a depolarization, which was highly dependent of the presence of sodium in the medium. 20 µM of nifedipine induced a depolarization of 6.9±0.8 mV (n=21). Dose response curve with nifedipine gave an EC50 in the 10µM range. We conclude that MDCK cells exhibit a dihydropyridine-activated cationic channel. Experiments are now undertaken to precise the nature of this permeability.
CNRS UMR 6548 Faculté des Sciences UNSA, NICE France / * Depart. de Fisiologia, CINVESTAV del IPN, Mexico, Mexico, D.F
THE CNS CATECHOLAMINERGIC CELL LINE CAD EXPRESSES TTX-SENSITIVE VOLTAGE-GATED SODIUM CHANNELS
Harvey V., Smith K., Garner C., McDonald R.L.
Voltage-gated sodium channels (VGSCs) play a central role in signal transmission in the central nervous system (CNS). Mutations of the alpha-subunits of neuronal VGSCs are implicated in several disorders including generalised epilepsy with febrile seizures (GEFS+) and familial autism. The molecular physiology of VGSCs in the catecholaminergic CNS cell line CAD has been investigated using the whole-cell patch-clamp technique and reverse transcription-polymerase chain reaction (RT-PCR).
Transient inward currents were evoked by 15ms step depolarisations from a holding potential of –60mV, following a 100ms hyperpolarising prepulse to –100mV. The transient inward current activated at -37 +/- 1mV, peaked at 0 +/- 1mV and measured 57.4 +/- 6.5pA/pF (n=13). The reversal potential (Erev), 49 +/- 2mV (n=13) was similar to the theoretical Erev for a Na+ -selective conductance under these conditions. Bath perfusion with 300nM tetrodotoxin (TTX) reduced the transient inward current at all test potentials; where currents measured at 0mV were reduced from 46.4 +/- 3.0 to 1.73 +/- 0.3pA/pF (p<0.001; 96.4% +/- 0.5; n=5). Following the identification of VGSC current, the candidate subtypes were further investigated. Consequently, the expression of TTX-sensitive VGSC mRNA was examined using RT-PCR. Total RNA was isolated from CAD cells using the Promega SV Total RNA isolation system. Upstream and downstream oligonucleotides specific for the alpha-subunits encoding Nav1.1, 1.2, 1.3, 1.6 & 1.7, were designed to anneal in different exons. Using the Promega Access RT-PCR system, only the alpha-subunits encoding Nav1.2, 1.3, 1.6 & 1.7 transcripts of the correct size were detected.
These results show that CAD cells express functional TTX-sensitive VGSCs and may provide a unique tool for their further study.
University of Huddersfield, Huddersfield, UK
S3 Environmental physiology
TRANSPORT AND ENERGETICS OF EXTREMOPHILES
Konings W.N., Albers S.V., Koning S., Driessen A.J.M.
The ion permeability of cytoplasmic membranes play crucial roles in the bioenergetics of micro-organisms. The proton and sodium-ion permeabilities were measured in liposomes prepared from lipids isolated from psychrophilic, mesophilic, thermophilic and hyperthermophilic bacteria and archaea and from halophilic archaea. In all membranes the proton and sodium-ion permeabilities increased with temperature. Membranes from psychrophilic and mesophilic bacteria and from mesophilic,(hyper)thermophilic and halophilic archaea have similar proton permeabilities at their respective growth temperatures. These observations indicate that micro-organisms are capable of adjusting the lipid composition of their membranes in order to maintain the proton permeabilities constant (homo-proton permeability adaptation).
Thermophilic bacteria are an exception in this respect and are unable to maintain a constant proton permeability at their high growth temperatures. As a result their membranes are very leaky for protons and a significant proton motive force cannot be build up in these organisms. The sodium-ion permeabilities were found to be very low and similar in all micro-organisms studied. Thermophilic bacteria make use of this low sodium-ion permeabilities to generate a sodium motive force which is subsequently used as a driving force for energy-requiring membrane processes such as secondary solute uptake systems.
In thermophilic arcaea such as Pyrococcus furiosus and Sulfolobus solfataricus several binding-protein dependent ABC-transporters have been found to catalyze the transport of their carbon and energy sources. These binding proteins have high affinity for their substrates. Interestingly, P.furiosus possesses ABC transporters that catalyze the uptake of oligomers of maltose and cellobiose.
Molecular Microbiology, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands
PHYSIOLOGICAL ADAPTATIONS OF ANIMALS TO CLIMATIC CHANGES AND LIMITATIONS IN RESOURCES
Le Maho Y.
Evidence is now accumulating, suggesting that the climate of past decades may be anomalous compared with earlier climatic variations. Numerous models moreover predict an increase in these climate anomalies, which may induce limitations in resources.
Investigating how animals may face climatic conditions is therefore an important issue. However, until recently, there was a severe limitation in our ability to get detailed information on free-ranging animals. Thanks to the prodigious progress in micro-electronics and computers it is now possible to obtain physiological and behavioural data of animals diving far at sea, wandering into the oceans, flying over deserts or mountains. Not only we are now able to get data on the biology of animals under natural conditions, but through ultra-miniaturized instruments animals deliver us detailed information on their environment.
This lecture will therefore provide examples of new discoveries in the quickly developing field of ecophysiology.
Centre d’Ecologie et Physiologie Energétiques (UPR CNRS 9010), Strasbourg, FRANCE
DNA REPAIR CAPACITY OF IRRADIATED HUMAN LYMPHOCYTES EVALUATED BY COMET ASSAY
Cardile V., Renis M., Scifo C., Bellia M., Lombardo L., Perciavalle V.
DNA is frequently damaged by endogenous and environmental agents, which provoke cellular deleterious consequences. Cells, however, have evolved sophisticated systems in response to DNA damage which constitute crucial defence systems against cytotoxicity, mutagenesis and carcinogenesis induced by DNA damaging agents. Therefore, DNA repair is regarded as one of the essential events in all life forms. Ionizing radiation, interesting for its environmental and clinical implications, is a potent inducer of DNA damage because it causes single- and double-strand breaks, alkali-labile sites, base damage, and crosslinks. This study was aimed to determine whether the alkaline COMET assay (single cell gel electrophoresis) can be used to evaluate DNA repair after damage induced by ionizing radiation. Resting peripheral blood lymphocytes, isolated from fresh buffy coats of 8 healthy blood volunteers by means of Lymphoprep gradient centrifugation, were analysed after exposition to four different doses (0.5, 1.0, 1.5 or 2.0 Gy) of X-ray radiations, followed by incubation at 37°C in a mixture 5% CO2/95% air, for 2, 4, 8, 24, 48 or 72 h. For each incubation time, exposed and control (unexposed) lymphocytes were scored by fluorescence microscopy using Scion Image software, and COMET tail length, percentage of fragmented DNA (TDNA), and tail moment, expressing the product of the tail/head and TDNA, were measured. The results indicated that irradiated cells, examined 2 or 4 h after the treatment, showed a dose-dependent increase of DNA single and double-strand breaks. DNA repair was observed in 1.5 and 2.0 Gy treated lymphocytes examined 8 and 24 h after the treatment, compared to controls. Both treated and untreated lymphocytes showed drastic DNA damage 48 and 72 h after the treatment, because of their known short life time. Our results confirm COMET assay as a rapid, simple and sensitive technique for visualizing and measuring DNA damage leading to strand breakage in individual cells.
Department of Physiological Sciences, University of Catania, Catania, Italy
CARDIAC NATRIURETIC PEPTIDE AND URINE FLOW IN HYPOXIC TROUT
Tervonen V., Vuolteenaho O.*, Nikinmaa M.
When acutely exposed to hypoxia, vertebrates show a rapid hemoconcentration resulting from a decrease in plasma volume and release of erythrocytes from the spleen. Reduced plasma volume is at least partly due to increased renal water excretion, a common transient response to acute hypoxia in vertebrates. One of the potential endocrine mechanisms mediating the diuretic response in hypoxia is cardiac natriuretic peptides. These peptide hormones have potent diuretic and natriuretic effects and they play an important role in modulating intravascular volume homeostasis. To elucidate the role of cardiac natriuretic peptides in hypoxic diuretic response, we used rainbow trout (Oncorhynchus mykiss) and a recently cloned salmon cardiac natriuretic peptide (sCP) as a model. In aquatic environment large variations in oxygen tension may occur and thus fishes encounter hypoxia regularly. To study the effect of hypoxia on cardiac natriuretic peptide plasma levels and urine flow, adult freshwater trout, kept in 12 ºC, were cannulated in the dorsal and ventral aorta and in the urinary bladder. The trout were exposed to hypoxia (3 mg O2/l) for three hours. The urine flow increased almost immediately in trout exposed to hypoxia and remained at an elevated level for the following two hours. Simultaneously, the plasma immunoreactive sCP (ir-sCP) concentration showed a significant increase in both the ventral and the dorsal aorta. Thus, in trout, an increased plasma ir-sCP level occurs as an immediate response to hypoxia and increase coincides with hypoxic diuretic response. Supported by the Academy of Finland.
Univ. of Turku, Laboratory of Animal Physiology, Turku, Finland, *Univ. of Oulu, Dept of Physiology, Oulu, Finland
MICROARRAY ANALYSIS OF CIRCADIAN GENE EXPRESSION IN MOUSE LIVER
Lacoche S., Gréchez-Cassiau A., Teboul M., Azmi S., Laudet V., Taneja R., Delaunay F.
Circadian rhythms in physiology are observed in most living organisms from cyanobacteria to humans. These rhythms are generated by a self sustained endogenous clock that is reset by the light/dark cycle and which in turn regulates rhythmically downstream pathways. Biochemical and genetic studies have established that a small group of genes termed clock genes generates a molecular oscillator through a transcriptonnal/translational feedback loop mechanism. Circadian oscillators are present not only in the suprachiasmatic nuclei of the hypothalamus but also in most peripheral organs. To understand how peripheral circadian oscillators regulate rhythmic physiological processes we have analysed circadian gene expression in mouse liver using high density oligonucleotide microarrays. We have identified ~ 250 rhythmic transcripts that regulate a wide variety of biological processes including metabolism, transcription, transport and signal transduction. Peaks of expression are found at all circadian times yet with a majority of transcripts peaking at dusk and dawn. Several transcriptonnal regulators have been identified suggesting that clock-controlled gene expression is mainly indirect in mammals. We show that the bHLH transcriptonal repressor Stra13 is rhythmically expressed in most peripheral tissues and that its promoter is regulated by the clock gene products CLOCK and BMAL. These data show that circadian gene expression is extensive in liver and suggest that Stra13 may be an important link between peripheral oscillators and physiological outputs.
Université de Nice-Sophia Antipolis UMR CNRS 6078, Villefranche/Mer, France
EFFECT OF AEROBIC TRAINING ASSOCIATED WITH HYPOXIC EXPOSITION DURING SLEEP ON ANTIOXIDANT CAPACITY
Pialoux V., Mounier R., Gueux E., Mazur A., Rayssiguier Y., Coudert J., Fellmann N.
The aim was to determine, on high-level nordic skiers, the impact of physical training associated with hypoxia during sleep on the antioxidant capacity evaluated by two direct methods and on the production of a lipoperoxidation (malondialdehyde, MDA) marker following an in vitro oxidation.
Eleven subjects were divided in 2 groups. The first group (H) (n=6) trained at low altitude (1,100m) and slept in normobaric hypoxic room during 3 weeks: simulating 2,500 m during the first week, 3,000 m the second week and 3,500 m the third week, and the second group (N) (n=5) were submitted to the same training but slept at 1,100 m of altitude. Venous blood samples were collected in pre-training state (I), immediately at the end of training session (II) and 2 weeks after (III).
Plasma Trolox equivalent antioxidant capacity (TEAC), ferric reactive antioxidant potential (FRAP) were analysed in I, II and III periods. The difference between MDA induced in vitro (MDA-i) and non-induced (MDA-ni), considered as an indirect evaluation of plasma antioxidant capacity, was measured in I and III. TEAC and FRAP were decreased by training (I vs II) in both groups (H=-21%, p=0.03, N=-13%, p=0.04 for TEAC and H=-20%, p=0.01, N=-17%, p=0.03 for FRAP). Only TEAC returned to its basal value in III whereas FRAP values remained significantly lower than in I. For each periods, the FRAP and TEAC values were highly correlated (r=0.85, r=0.63 and r = 0.80 for I, II and III respectively). The difference between MDA-i and MDA-ni were higher in III than in I (H=+70%, p=0.04 and N=+59%, p=0.006). No significant differences were found between H and N for each methods.
Regardless the methods used, an intense aerobic training was responsible for an antioxidant capacity decrease, which persisted after 2 weeks of recovery and the associated hypoxia did not worsen the deficiency.
This study was funded by the Olympic Committee and the "Direction Régionale de la Jeunesse et des Sports Auvergne" - France
Laboratoire de Biologie-Physiologie du Sport, Faculté de Médecine, Clermont-Ferrand, France
ROLE FOR Na+/H+ EXCHANGER 1 IN XENOBIOTIC-INDUCED APOPTOSIS IN LIVER EPITHELIAL CELLS
Huc L., Sparfel L., Rissel M., Fardel O., Lagadic-Gossmann D.
Polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene B(a)P, are ubiquitous environmental pollutants to which humans are commonly exposed. They are responsible for important carcinogenic and apoptotic effects, whose mechanisms are still poorly understood. Among these mechanisms, perturbations of H+ homeostasis may be involved. This work has been carried out in order to test the effects of B(a)P (50nM) on pHi in rat liver F258 epithelial cell line, using carboxy-SNARF-1 as pH-sensitive fluorophore. B(a)P induced biphasic pHi changes, with first an alkalinization (at 48h) followed by an acidification (at 72h). Determinations of pHi recovery following an acid load showed an increase of acid efflux at 48h. By using cariporide, a specific Na+/ H+ exchanger 1 inhibitor, we demonstrated that NHE1 was activated upon B(a)P treatment and was responsible for pHi changes at 48h. The alpha-naphtoflavone (NF), a CYP1A1 inhibitor, as well as the antioxidant thiourea prevented any pHi variation induced by B(a)P, thus indicating a dependence of NHE1 activation upon reactive oxygen species (ROS) produced during B(a)P metabolism. When analysing B(a)P-induced apoptosis, we found that cariporide significantly reduced both DNA fragmentation and caspase-3 like activity. Using flow cytometry and the fluoroprobe dihydroethidium, we further showed that NHE1-dependent early alkalinization affected the mitochondrial ROS production detected during the apoptotic cascade.Altogether, our results suggest that B(a)P, via metabolism-dependent ROS production, induces an early activation of NHE1, thus leading to an alkalinization that might play a significant role in the subsequent induction of mitochondria-dependent apoptosis.
INSERM U456, Univ Rennes 1, Fac de pharmacie, 2 av Pr L Bernard, 35043 Rennes cedex, FRANCE
MOLECULAR MECHANISMS OF GENETIC ADAPTATION TO XENOBIOTIC COMPOUNDS
Organisms are permanently exposed to the environment and their response to this environment will determine their survival (short term) or their evolutionary success (long term). Adaptation to xenobiotics is a specific case of adaptation to the myriad of chemicals of natural origin, but it is of particular importance to human well being, because our food or our health is often protected or restored by xenobiotics - pesticides, drugs and antibiotics. This lecture will focus on resistance as an example of genetic adaptation to xenobiotics. The widespread use of insecticides has amounted to a large scale "experiment' in natural selection of insects by chemicals which are often of toxicological importance to humans as well. The biochemical and physiological mechanisms of resistance can be categorized as target site insensitivity, increased metabolic detoxification and sequestration or lowered availability of the insecticide. At the genetic level, the mutations in receptors, transporters or enzymes may be classified into those that alter binding or catalysis by structural changes, up regulation including gene amplification, or down regulation including gene disruption or silencing. Regulation can be altered either by cis- or trans-acting control of expression. Genomic approaches greatly acelerate the discovery of resistance mutations. In several cases, the selection of a precisely homologous mutation has been observed in different species, indicating that resistance is an extreme case of genetic adaptation.
INRA, Antibes, France
CIRCADIAN CLOCKS: FROM GENE EXPRESSION TO PHYSIOLOGY AND DISEASE
Schibler U., Gachon F., Ripperger J., Brown S.A., Gos P., Le-Minh N., Preitner N.
Circadian pacemakers were originally believed to exist only in a few specialized cell types, such as neurons of the suprachiasmatic nucleus (SCN). However, in recent years, this view has been challenged by the discovery that circadian clocks may exist in most peripheral cell types, and even in immortalized tissue culture cells. Nevertheless, these subsidiary oscillators have to be synchronized periodically by the central pacemaker in the SCN. Our studies suggest that this is accomplished mostly via indirect ways. In fact, feeding time is the most dominant Zeitgeber for peripheral clocks. Thus, the SCN entrains the phase of circadian gene expression and physiology in the periphery primarily by setting the phase of rest-activity cycles, which in turn determines the time of feeding. In addition, body temperature rhythms and cyclically secreted hormones also participate in the synchronization of peripheral clocks.
We have explored biochemical and genetic approaches to identify and study transcriptional regulatory proteins that translate the oscillations generated by the molecular clockwork into overt rhythms in physiology and behavior. TEF, HLF, and DBP, the three members of the PAR bZip protein family, strongly oscillate in liver and other peripheral tissues and thereby regulate the cyclic expression of several target genes (e.g. cytochrome P450 enzymes). In brain regions other than the SCN, however, the levels of these transcription factors fluctuate with only a small amplitude, and never fall below 30% to 50% of maximal circadian values. Our genetic loss-of-function experiments may offer a plausible explanation for why high amplitude cycles of Dbp, Tef, and Hlf gene expression cannot be tolerated in the brain. In fact, these transcription factors protect the mice from lethal seizure attacks (epilepsies) and thus have to be present throughout the day in the central nervous system.
Department of Molecular Biology, Sciences II, University of Geneva - Switzerland
THE EFFECTS OF EXOGENOUS PROSTAGLANDINS AND CYCLOOXYGENASE INHIBITOR ON APOPTOSIS IN RAT HEPATOCYTES
Korniychuk G.M., Khabatyuk N.G., Makogon N.V., Alexeyeva I.N.
In addition to the fact that hepatocytes mainly take part in degradation of eicosanoids, they also produce small amounts of prostaglandins (PG) I2, E2, F2α, TxA2, which act primarily in cell-to-cell communications. The aim of the study was to investigate the influence of cyclooxygenase inhibitor (acetylsalicylic acid – AA) and exogenous PGE2 and PGF2α on apoptosis and necrosis in isolated rat hepatocytes under normal and pathological conditions, by method of fluorescent light microscopy after double cell staining with Hoechst 33342 and propidium iodide. It has been demonstrated, that under normal conditions neither AA nor exogenous PGE2 and PGF2α in a wide range of concentrations (0,1 microM - 3 microM) did not significantly change the ratio of vital, necrotic and apoptotic cells. Carbon tetrachloride (CCl4) administration (10 mM) caused a decrease in amount of vital cells and an increase of necrotic and apoptotic cells. Treatment of hepatocytes with AA (20 microM) before CCl4 adding led to a decrease in apoptotic cells count, whereas treatment with exogenous PGE2 and PGF2α (0,9 microM and 3 microM) on the contrary, increased the number of apoptotic cells. These results suggest that cyclooxygenase pathway of arachidonic acid metabolism is an important modulator of hepatocyte viability and death.
Bogomoletz Institute of Physiology of NAS Ukraine, Kyiv
ANTIOXIDANT EFFECT OF A, E, C VITAMINS, TOPICALLY ADMINISTERED AFTER UVB RADIATION EXPOSURE
Ultraviolet radiation lead to reactive oxygen species occurrence in skin and decrease local antioxidant capacity.
We intended to highlight the antioxidant role of A,C, E vitamins, topically administered, after UVB exposure (2,4 J/cm2). Vitamins were applied on skin rats Wistar race, on a surface of 2cm2, previously shaved. We assessed lipid peroxides, total SH groups and non-proteic thiol from tegument at one hour, respectively 24 hours after exposure.
Topically administration of A and C vitamins significantly decreased the lipoperoxidation processes (p<0.01) after one and 24 hours, while E vitamin only after 24 hours. Total SH groups significantly raised at one hour and 24 hours after alpha-tocopherol treatment. A vitamin was efficient after 24 hours (p<0.001). Topically treatment with C vitamin had a defensive effect proved through the increase of total SH groups, one hour after exposure.
Our data demonstrates that topically treatment with vitamins decreases lipoperoxidation reactions due to UVB and leads to total SH groups and non-proteic thiol regeneration in skin.
University of Medicine and Pharmacy "Iuliu Hatieganu" – Cluj-Napoca, Romania
CORRECTION OF METABOLIC DISORDERS AT HYPOXIA BY NEW PHARMACOLOGICAL PREPARATIONS
Gonchar O., Klyuchko E., Seredenko M., Oliynyk B.
We studied substances that may be potential medicines for hypoxia disorders treatment: yackton and sufan - derivatives of succinic acid, and splenoside – non- protein factor of spleen with nucleoside complex as active base. Investigations were carried on homogenates, cytosol and mitochondria fractions of liver, heart, lungs, brain tissues of Wistar rats during acute hypoxias: hypoxic, hemic (after the injection of sodium nitrite (6 mg / 100 g/rat weight) and circulatory hypoxia (after bleeding – 2,5 ml / 100 g/rat weight). During the hypoxic syndrome development we registered an increase of lipid peroxidative oxidation (LPO) in all studied tissues, disorders in enzymatic and non- enzymatic antioxidant system activities, acidosis development, depression of electron transport and mitochondrion functions of energy synthesis. Degree of expression for these processes depended from the type of hypoxia and tissue specificity.
Preliminary administration of any preparation (before the extreme influence)– yackton, sufan or splenoside caused the decrease of LPO, increase of activity of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase as well as increase of reduced glutathione content in comparison with hypoxia state. NAD/NADH rate increased and decreased lactate/ piruvate rate and lactate dehydrogenase activation.
After the splenoside injection we registered an activation of glucoso-6-phosphatedehydrogenase (with the slight changes in succinate dehydrogenase activity (P<0,5) that evidences about the comparative priority of pentose- phosphate pathway. After the yackton injection in hypoxia conditions we registered in mitochondria an increase of succinate dehydrogenase activity (P<0,01) that leaded to the reduction of electron transport and recovery of energy synthesis functions in mitochondria. So, all studied preparations demonstrated antioxidant effect at hypoxia and may be potential medicines.
Bogomoletz Institute of Physiology, National Academy of Science, Kiev, Ukraine