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S1-5

THE MODE OF ACTION OF POTASSIUM ON Ca2+ SIGNALLING AND ALDOSTERONE PRODUCTION IN GLOMERULOSA CELLS

Spät A., Koncz P., Makara J.K., Pitter J.G.

Potassium balance is controlled by aldosterone, secreted by adrenal glomerulosa cells. These cells are uniquely sensitive to extracellular K+ concentration. Hyperkalaemia depolarizes the glomerulosa cell, thus activating voltage-dependent Ca2+ channels. Rat glomerulosa cells display Ca2+ signal in response to raising [K+] from the control 3.6 mM by as little as 0.5 mM and at 4.6 mM aldosterone production rate is already doubled. Resting membrane potential is ~ -80 mV and the activation threshold of T-type Ca2+ channels is between –70 and –80 mV. Activation of these channels by K+ in rat glomerulosa cells is attributed exclusively to depolarization (Lotshaw: Mol. cell. Endocrin. 2001) and increased hormone secretion is attributed to Ca2+-induced transport of cholesterol into mitochondria. We studied both the generation and action of Ca2+ signal. Microfluorimetry, digital imaging and patch-clamp techniques were applied on primary cultures of rat glomerulosa cells.

Swelling induced by hyposmosis (measured on calcein-loaded cells) shifts the activation threshold of T-type channels to more negative potentials, enhances K+-induced Ca2+ signal as well as aldosterone production. Swelling can be induced also by raising [K+] (from 3.6 to 5 mM) and this swelling coincides with a second phase of elevation of cytoplasmic [Ca2+]. Prevention of K+-induced swelling with appropriate hyperosmosis attenuates the Ca2+ signal. Therefore, swelling seems to amplify the action of K+-induced depolarization on Ca2+ channels.

Physiological increase in [K+] evokes elevated mitochondrial NAD(P)H level in a Ca2+-dependent manner. The reoxidation of NAD(P)H depends on the rate of steroid synthesis. Low submicromolar elevation of cytoplasmic [Ca2+] raises mitochondrial [Ca2+] and the ensuing reduction of pyridine nucleotides may support increased steroid production.

Supported by OTKA (T-032270, TS-040865) and ETT (28/2000).

Dept. of Physiology, Semmelweis University, Budapest - Hungary

POSTER SESSION

P01-01

INHIBITION OF RETICULAR CALCIUM UPTAKE ALTERS THE EFFECT OF PASSIVE TENSION ON RAT AORTA CONTRACTION

Serban I.L., Serban D.N., Petrescu G

The length-tension relationship, as a determinant of muscle contraction, is highly variable among smooth muscles and the mechanisms remain elusive. We investigated the effect of passive tension on the contractile response of the de-endothelised isolated rat aorta and the influence of reticular calcium pump inhibitors therein. De-endothelised aorta rings (2 mm wide) from male adult Wistar rats were studied in isometric conditions; oxygenated saline solution (HCO3- buffer, pH 7.2-7.4), at 37 °C. Each ring equilibrated for 2 h under a passive tension of 2 g, then was contracted by 0.01 mM phenylephrine (PE); results expressed as % active tension of this reference value in each preparation (mean ± S.E.M.; n = 6 in all series). All rings were randomly subjected to 1 h re-equilibration under 0.5, 2, or 3 g, then a concentration-effect curve for either PE alone or in the presence of 100 nM thapsigargin (THAP) or 0.01 mM cyclopiazonic acid (CPA), to eliminate the reticular pump influence upon cytosolic calcium signals. The curve is shifted to the right at the lower passive tension, while effects are significantly increased by the higher resting tension only for low PE doses. In agreement with previous findings, elevation of cytosolic calcium by THAP or CPA enhances contractions induced by low and moderate PE concentrations. We found this effect to be prominent at low passive tension and weaker with higher stretch. Reticular pump inhibition enhances aortic contraction presumably by elimination of the buffering effect of reticular calcium uptake. Beside other mechanisms investigated so far, the potentiating effect of passive tension could involve inhibition of the reticular calcium pump.

University of Medicine and Pharmacy 'Gr. T. Popa' – Iasi - Romania

P01-02

PLASMA MEMBRANE AND NUCLEAR ENVELOPE NPY AND Y1 RECEPTOR IN HUMAN ENDOCARDIAL ENDOTHELIAL CELLS

Perreault C., Jacques D.

Using 3D confocal microscopy and immunofluorescence, we tested the hypothesis that endocardial endothelial cells do possess NPY and NPY receptors and that activation of these receptors may modulate cytosolic and nuclear free calcium. Our results showed that effectively, NPY is present in human endocardial endothelial cells at both the cytosolic and nuclear levels. This peptide was found to be secreted by these cells upon sustained increase of intracellular calcium. In addition, our results showed that Y1 receptors are present all through the cell including the nuclear membranes. Activation of NPY receptors induced a dose-dependent increase of both cytosolic and nuclear calcium. This effect of NPY was largely due to activation of Y1 receptors. In conclusion, our results demonstrate that human endocardial endothelial cells do secrete NPY via a calcium-dependent mechanism. In addition, NPY and its receptors modulate intracellular calcium, thus affecting excitation-secretion coupling of endocardial endothelial cells. This work was supported by the Canadian Institutes of Health Research.

Department of anatomy and cell biology, Faculty of medicine, University of Sherbrooke, Sherbrooke, QC, J1H 5N4, Canada

P01-03

MODULATION OF INTRACELLULAR CALCIUM BY SARCOLEMMA AND NUCLEAR MEMBRANES Ang II AT1 RECEPTORS.

Nader M., Bkaily G.

The objective of the study was to verify if human (h) AngII type-1 receptor (hAT1R) undergoes transcellular trafficking in human aortic vascular smooth muscle cells (hVSMCs)and if overexpression of this receptor modulates cytosolic and nuclear free calcium. Three-dimensional (3D) confocal microscopy was used to monitor hAT1R-GFP (green fluorescence protein fusion. Using 3-D imaging technique, hAT1Rs were localized at the sarcolemma, in the cytosol and in the nuclear compartments. Stimulation of sarcolemma membrane hAT1Rs by Ang II induced internalization and nuclear translocation of this type of receptor. The internalization of hAT1Rs was found to be mediated via clatherin-coated pits and vesicles pathway. The internalization and translocation of hAT1Rs was associated with a de novo-synthesis of this type of receptor. Overexpression of hAT1Rs induced a decrease of both cytosolic and nuclear free Ca2+. In conclusion, our results susggest that hAT1Rs are the predominant type of Ang II receptors in aortic hVSMCs and are present in the sarcolemma, the cytosolic and nuclear compartments. Overexpression of these receptors modulate cytosolic and nuclear free calcium. This work is supported by a grant from the Canadian Institute of Health Research (CIHR).

Department of Anatomy and Cell Biology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada

P01-04

REGULATION OF GABA RELEASE BY CALCIUM TRANSIENTS AT A SINGLE HIPPOCAMPAL TERMINAL

Fedulova S.A., Verkhratsky A.N., Veselovsky N.S.

We correlated dynamic changes in free Ca2+ concentration ([Ca2+]i) within single presynaptic terminal of cultured hippocampal neurones with postsynaptic GABA-mediated currents. For this purpose local changes in [Ca2+]i and evoked inhibitory postsynaptic currents (eIPSCs) were recorded simultaneously using Fura-2 fluorescence and whole-cell patch-clamp. The Ca2+ signals and eIPSCs were evoked by direct extracellular electrical stimulation of a single presynaptic terminal by short depolarizing pulses. All experiments were performed in 0.25 *M TTX-containing solution to suppress action potential generation.

The presynaptic Ca2+ transient was changed by varying the amplitude of the extracellular stimulating pulses. The probability of release event, estimated for the each stimulation strength, changed from 0 to 1. The release probability reached P = 1 since the Ca2+ signals attained maximal value and remained at this level at higher stimulation strength despite the decrease in the amplitude of the Ca2+ transients. In the range of stimulating amplitudes, where release probability was P < 1, a Ca2+ signal of the same amplitude could result in either failure of the postsynaptic response or an IPSC of any random amplitude.

Linear gradual increase in stimulation amplitude (Vstim) resulted in a bell-shaped dependence of the averaged amplitudes of Ca2+ signals and corresponding averaged amplitudes of eIPSCs. Aanalysis of eIPSC demonstrated that decrease in mean eIPSC amplitude as well as reduction in quantal content of release resulted from a reduction in the probability of multivesicular release i.e. in the disappearance of failures and decrease in individual eIPSC amplitude. Ca2+ signals of similar amplitude resulted in both random and non-random release characteristics. We concluded that depolarization-induced [Ca2+]i elevation within the terminal is necessary but not sufficient for activation of vesicular release.

Bogomoletz Institute of Physiology, National Academy of Science & School of Biological Sciences, Manchester University

P01-05

CILIARY NEUROTROPHIC FACTOR RAPIDLY INHIBITS VOLTAGE-GATED SODIUM CHANNELS IN SKELETAL MUSCLE

Talon S., Metges-Giroux M.-A., Pennec J.-P., Gioux M., Léoty C.

The ciliary neurotrophic factor (CNTF) is known to exert long-term myotrophic effects, but it is not yet evidenced if this cytokine could also induce a rapid biological response in skeletal muscles. The present in vitro study brings up the possibility that CNTF could affect the nerve-muscle coupling implied in the rapid triggering of muscle fibre contraction, particularly by influencing sodium channel activity. Therefore, we investigated the effects of an external 10-min application of 2ng.ml-1 CNTF on macroscopic sodium current (INa) of rat native fast-twitch skeletal muscle (flexor digitorum brevis, FDB) by using a cell-attached macro-patch technique. The fibres were isolated by enzymatic dissociation then cultured in 35mm Petri dishes for the experiments duration. Compared to control conditions, CNTF rapidly reduced the peak value of INa by 30.4 ± 6.3% (n=10, p<0.005) with a voltage step depolarising the path membrane from –100 to –10mV. No significant change was observed in activation and inactivation kinetics. Normalized current-voltage (I/V) curves in the presence and in absence of CNTF were superimposed, indicating the lack of CNTF effect on the voltage-dependence of sodium channel activation in FDB muscles. In the opposite, the relative INa blockade induced by CNTF was accompanied by a shift of inactivation curves to more negative potentials, the shift in half-maximal potential being DVH2/2 = -5.7 ± 1.3 mV (n=10, p<0.005) and DVs1/2 = -8.8 ± 2.1 mV (n=5, p<0.005) for the steady-state fast and slow inactivation respectively. These results suggest that CNTF can rapidly induce a decrease in skeletal muscle sodium currents, probably through an intracellular mechanism different from the well-characterized JAK/STAT pathway. The present study would then contribute to better understand the physiological role of endogenous CNTF.

CNRS UMR 6018 Développement et Physiologie des structures contractiles, 2 rue de la Houssinière Nantes 44322 - France

P01-06

IMPROVEMENT OF CELL SURVIVAL AND CALCIUM SIGNALING PROPERTIES IN MUSCLE CELLS BY BMD MINIDYSTROPHIN

Vandebrouck A., Constantin B., Marchand E., Cantereau A., Basset O., Pelletier F., Claudepierre M.C., Braun S., Ruegg U., Raymond G., Cognard C.

Defective expression of dystrophin in muscle cells is the primary feature of Duchenne Muscular Dystrophy (DMD). Absence of 427kDa dystrophin is accompanied with a chronic elevation in intracellular calcium concentration, leading to fiber necrosis. However, direct evidence that dystrophin can control calcium handling in muscle cells has not yet been proved. Mutations of the dystrophin gene lead to DMD or the milder Becker Muscular Dystrophy (BMD) which is associated with the expression of a truncated 229kDa protein. A 6.3 kb minidystrophin cDNA has been cloned from an asymptomatic BMD patient. Its size is sufficiently small to be accommodated by current retroviral vector systems and it has been successfully expressed in the myogenic Sol8 dystrophin-deficient cell line with accumulation of the minidystrophin at the sarcolemma. Forced expression of the minidystrophin reactivates appropriate sarcolemmal expression of dystrophin-associated proteins, and leads to a decrease in cell death. We have measured the calcium influx with a cytophotometer and the fluorescent calcium probe Indo-1 as well as the intramitochondrial calcium by transfection with Aequorin, a luminescent calcium probe targeted to mitochondria. Minidystrophin forced expression decreases the amplitude of cytosolic calcium transients induced by membrane depolarisation. We have observed that depletion of sarcoplasmic reticulum leads to a store-operated calcium influx, which is less sustained in myotubes expressing minidystrophin. These store-operated calcium influx also lead to calcium entry into mitochondria, a major calcium buffer of muscle cells. These intramitochondrial entries are also shorten in myotubes expressing minidystrophin. We propose that dystrophin could regulate sarcolemmal calcium channels likely through linkage with Alpha-syntrophin, but also on intracellular calcium channels behaviour.

LBSC UMR CNRS/Université de Poitiers 6558, PBS 86022 Poitiers cedex, France

P01-07

CHANGES IN SR CA-ATPASE EXPRESSION AND CYCLOPIAZONIC ACID SENSITIVITY IN EDL MUSCLE FROM MDX MICE

Divet A., Lompré A.-M., Huchet-Cadiou C.

Duchenne muscular dystrophy (DMD) results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane in skeletal muscle. Although increased sarcolemmal Ca2+ influx in dystrophic muscle is proposed as an early event in DMD pathogenesis, Ca2+ handling mechanisms are not clearly understood. In this study, we investigated the sarcoplasmic reticulum (SR) properties in fast- (edl) and slow- (soleus) twitch muscles from 4-week-old control and mdx (C57BL/10mdx) mice, an animal model for DMD. The results show that in saponin skinned fibres, where the SR was functional, the Ca2+ uptake was slower in mdx muscles while the maximal Ca2+ loading capacity was maintained. In both types of mdx muscles, the time to load the SR was significantly increased but this was more pronounced in soleus fibres. Cyclopiazonic acid (CPA), an inhibitor of the SR Ca2+-ATPase, induces a decrease in the Ca2+ uptake and the CPA sensitivity was decreased by 50% in mdx edl skinned fibres (control: IC50=10.1±1.7 µM CPA, mdx: IC50=20.2±1.7 µM CPA; n=8). In SR vesicles, the Ca2+-ATPase activity and CPA sensitivity was not affected by the dystrophic process in both types of muscles. The expression of the slow Ca2+-ATPase isoform (SERCA2a) at the mRNA and protein level was significantly increased in mdx edl muscle (SERCA1/SERCA2a mRNA: control=163.7±13.4, mdx=74.0±16.2; n=3). The expression of SERCA1 and SERCA2a was not modified in mdx soleus. The results show that the SR is involved in the abnormal Ca2+ homeostasis in both types of skeletal muscles. In mdx soleus muscle, the increase in SR Ca2+ loading time was not related to the Ca2+-ATPase function and expression, and then could be explained by an increase of the SR Ca2+ leakage. In mdx edl muscle, the decrease in CPA sensitivity in skinned fibres could be explained by the presence of the slow Ca2+-ATPase isoform. Then some SR properties of fast mdx muscles are similar to those observed in slow-twitch muscles.

CNRS UMR 6018 Developpement et Physiologie des structures contractiles, 2 rue de la Houssiniere Nantes 44322 France

P01-08

EXTRACELLULAR ATP-EVOKED CALCIUM FLUXES ON CULTURED MOUSE SKELETAL MUSCLE CELLS

Gönczi M., Szappanos H., Cseri J., Kovács L., Csernoch L.

Changes in intracellular calcium concentration ([Ca2+]i) were measured on cultured skeletal muscle cells from mice following the application of extracellular ATP. Established methods were used to determine the calcium flux, entering the myoplasmic space, from the measured changes in [Ca2+]i in order to assess the contribution of different sources of calcium in the formation of the ATP-evoked calcium transient. The resting [Ca2+]i decreased, from 99±4 (n=64) to 51±2 nM (n=104), while the transport capacity of the Ca-ATPase increased, from 107±10 to 596±36 µM/s, with differentiation. The calcium flux, evoked by 30-40 s long application of ATP, displayed an early peak (74±9 µM/s, n=35; in cells with less than 5 nuclei) and then declined to a quasi-maintained steady level (Sl; 17±3 µM/s). The peak was strongly reduced on depolarised cells and was completely missing on large myotubes. The removal of external calcium on the other hand, suppressed the quasi-steady level as well. Suramin, in a concentration of 10 µM, reduced Sl by 40±7% (n=7), while 300 µM suramin produced an almost complete block. Immunofluorescent labeling revealed the presence of both P2X and P2Y receptors in the surface membrane of these cells. The results demonstrate that extracellular ATP elevates [Ca2+]i through four, interconnected mechanisms: an influx through P2X receptors and voltage gated calcium channels, the release of calcium from intracellular stores through calcium-induced calcium release and IP3-sensitive channels.

University of Debrecen, Medical and Health Science Center, Medical School, Department of Physiology, Debrecen, Hungary

P01-09

FLUORESCENT IMAGING STUDIES OF NO PRODUCTION IN PANCREATIC ACINAR CELLS

Chvanov M., Gerasimenko O., Petersen O.H., Tepikin A.

We have analysed the synthesis of nitric oxide (NO) in acutely isolated pancreatic acinar cells using the fluorescent probes – membrane-permeable 4,5-diaminoflurescein diacetate (DAF-2 DA) and impermeable 3-amino-4-(N-methylamino)-2’,7’-difluorofluorescein (DAF-FM). Application of 10 µM acetylcholine (ACh) caused a rise in fluorescence in only 21 out of 107 cells loaded with DAF-2 DA. Much higher proportion of cells (14 of 20) displayed fluorescence changes to ACh application when the membrane-impermeable form has been used. In these experiments the indicator was delivered into the cytosol via patch pipette. The recordings of calcium-dependent chloride currents allowed us to correlate Ca2+ signals with changes of DAF-FM fluorescence. The DAF-FM responses were also seen when cells were stimulated with physiological (5 pM) and supramaximal (10 nM) doses of cholecystokinin (CCK), as well as at low doses (50 nM) of ACh. The DAF-FM responses to secretagogues were abolished by 300 µM melatonin and 300 µM carboxy-PTIO but remained intact in the presence of 300 µM uric acid, indicating specific detection of NO by DAF-FM. Addition 10 mM of calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) to patch pipette completely eliminated DAF-FM responses triggered by the secretagogues. These results suggest that pancreatic acinar cells produce NO during physiological activity and when stimulated with pathological doses of secretagogues.

The University of Liverpool, Liverpool, UINTED KINGDOM

P01-10

CHARACTERIZATION OF CALCIUM RELEASE EVENTS IN DYSTROPHIN DEFICIENT CELL LINES FROM SKELETAL MUSCLE

Balghi H., Sebille S. Cantereau A., Monory A., Tanguy S., Constantin B., Raymond G., Cognard C.

Skeletal muscle depolarization induces a massive release of stored calcium from the sarcoplasmic reticulum (SR) through the ryanodine receptors (RyR). Previous data suggest that an elevation in myoplasmic IP3 may be a secondary triggering signal for SR Ca2+ release during muscle activation. At rest, localized quantal Ca2+ release events (sparks) have been shown using laser scanning confocal fluorescence microscopy. Alterations of Ca2+ homeostasis are involved in Duchenne muscular dystrophy characterized by a lack of the dystrophin protein. The link between the absence of dystrophin and the Ca2+ mishandling remains unclear. Furthermore, there are only few data concerning a possible role of Ca2+ stored in the SR. The present study aims to characterize various events of Ca2+ release in a dystrophin deficient cell line (SolC1) and in SolD7, a stable dystrophin forced-expression derivative clone. Both spontaneous sparks and global Ca2+ release induced by perfusion of hyperpotassium (47 mM) depolarizing solutions were recorded. Using confocal microscopy, measurements of Ca2+ signals have been performed in myotubes loaded with the Ca2+ probe Fluo-4. SolC1 myotubes showed an intense sparks activity though SolD7 exhibited either low activity or not. During KCl perfusion, SolC1 myotubes show a slow kinetic both for Ca2+ increase and recovery. Although SolD7 myotubes displayed a recovery kinetic similar to SolC1 myotubes one, they showed a faster kinetic of Ca2+ release. Myotubes incubated with 2-APB (an inhibitor of IP3 receptors) showed a faster recovery kinetic than the control myotubes. These data suggest that IP3 could play a substantial role in Ca2+ release from SR in dystrophic cells. The characterization of both the expression and the organization of the calcium release channels (IP3 receptors) involved in this pathway has to be investigate more precisely in dystrophin-deficient models.

Laboratoires des Biomembranes et Signalisation Cellulaire, CNRS UMR 6558, Université de Poitiers, 86022 Poitiers cedex - France

P01-11

NAADP ACTIVATES A Ca2+ CURRENT WHICH IS DEPENDENT ON F-ACTIN CYTOSKELETON

Moccia F., Lim D., Nusco G.A., Ercolano E., Santella L.

NAADP is involved in the Ca2+ response observed at fertilization in the oocytes of several species, including starfish. In this study, we have employed Ca2+ imaging and the single-electrode voltage-clamp technique to investigate whether the NAADP-mediated Ca2+ entry discovered in our laboratory in starfish oocytes was mediated by a membrane current and whether the response to NAADP required an intact cytoskeleton. Uncaging of pre-injected NAADP evoked a cortical Ca2+ flash which was followed by the spreading of the wave to the remainder of the cell. No Ca2+ increase was detected in Ca2+-free sea water. Under voltage-clamp conditions, the photoliberation of NAADP activated an inward rectifying membrane current, which reversed at potentials more positive than +50 mV and was abolished by removal of Ca2+, but not of Na+. The current was affected by pre-incubation with verapamil, SK&F 96356 and thapsigargin, but not by pre-injection of heparin, 8-NH3-cADPr, or both antagonists. The membrane current and the Ca2+ wave were inhibited by latrunculin A and jasplakinolide, which depolymerize and stabilize actin cytoskeleton, respectively. These data offer the first demonstration that NAADP initiates a Ca2+ sweep by activating a Ca2+-permeable membrane current, which requires an intact F-actin cytoskeleton as other Ca2+-permeable currents, such as ICRAC and IARC.

Laboratory of Cell Biology, Stazione Zoologica "A. Dohrn", Naples I-80121, Italy

P01-12

LIPOXINE A4 STIMULATES A CALCIUM MOBILIZATION IN HUMAN AIRWAY EPITHELIAL CELL

Urbach V., Bonnans C.

Lipoxins (LX) are biologically active eicosanoids possessing anti-inflammatory properties. Using a calcium imaging system we investigated the effect of LXA4 on intracellular [Ca2+] of human bronchial epithelial cell. LXA4 produced a dose-dependent increase in [Ca2+]i followed by a recovery to basal values in primary culture of bronchial epithelial cell and in 16HBE14o- cells. The LXA4-induced [Ca2+]i increase was completely abolished by pertussis toxin (G protein inhibitor). The [Ca2+]i response was not affected by the removal of external [Ca2+] but inhibited by thapsigargin (Ca2+-ATPase inhibitor) treatment. Pre-treatment of the bronchial epithelial cells with either MDL hydrochloride (adenylate cyclase inhibitor) or Rp-cAMP (cAMP dependent protein kinase inhibitor) inhibited the Ca2+ response to LXA4. However the response was not affected by chelerytrine chloride (protein kinase C inhbitor) or montelukast (cysteinyl leukotriene receptor antagonist). The lipoxin A4 receptor mRNA was detected, by RT-PCR, in human bronchial epithelium. The functional consequence of the LXA4 effect on [Ca2+]i have been investigated on Cl- secretion, measured using the short-circuit techniques on 16HBE14o- monolayers grown on permeable filters. LXA4 produced a sustained stimulation of the Cl- secretion through 16HBE14o- monolayers, which was inhibited by BAPTA-AM, a chelator of Ca2+i. Taken together our results provided evidence for the stimulation of a [Ca2+]i increase by LXA4 through a mechanism involving its specific receptor and protein kinase A activation and resulting in a subsequent Ca2+-dependent Cl- secretion by human airway epithelial cells.

INSERM U454, Montpellier, France

P01-13

THE ROLE OF DEFECTIVE MITOCHONDRIA IN REGULATION OF Ca2+ INFLUX INTO OSTEOSARCOMA CELLS.

Szczepanowska J., Zablocki K., Duszynski J.

In non-excitable cells, the depletion of intracellular calcium stores localized in the lumen of endoplasmic reticulum leads to the opening of plasma membrane calcium channels termed SOC and finally, to an activation of Ca2+ influx into the cells. This regulatory phenomenon is also known as a capacitative Ca2+ entry. To explain the molecular mechanism of capacitative Ca2+ entry several hypotheses have been employed. One of them postulates that mitochondria can play an important role in the regulation of SOCs. In our study we examined a few osteosarcoma cell lines with mutated mitochondrial DNA (mtDNA). MtDNA contains 13 genes coding polypeptides required for the mitochondrial respiration and oxidative phosphorylation. In our investigations we used cells with different levels of heteroplasmy (mtDNA point mutation ATP6 gene, encoding subunit 6 of mitochondrial ATPase) and cells lacking mtDNA (without complete respiratory chain and ATPase). It has been shown that SOC activity was not reduced despite the impairment of mitochondrial energy status resulted from the mutations in mtDNA.

Moreover, we have examined the effect of thapsigargin-induced depletion of calcium stores localized in the ER and the CCCP – discharge of the mitochondrial electrochemical proton gradient on the mitochondrial and cytoskeletal organization and mitochondrial membrane potential in all the osteosarcoma cell lines. Confocal microscopy was used to visualize intracellular structures. Filamentous mitochondria were distributed along the cell body in the control cell line and in cells with only slight mitochondrial disorders. In the cells with low mitochondrial membrane potential due to high level of heteroplasmy as well as lack of mitochondrial mtDNA, mitochondria were small, and had round and cylindrical shape.

In conclusion, the results show that the regulation of capacitative calcium entry into osteosarcoma cells does not depend on the mitochondrial energy status.

Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland

P01-14

EFFECTS OF CHOLINERGIC BLOCKADE AND APAMIN ON RABBIT JEJUNUM MOTILITY AND ADRENERGIC INHIBITION

Martinoli L., Amico M.C., Ippoliti A., Romanelli L., Savini G., Tucci P., Valeri P.

Our aim was to investigate the influence of the cholinergic system and apamin-sensitive Ca2+-activated K+ channels on spontaneous contractions in rabbit jejunum and on the a1- and b-adrenoceptor-mediated inhibition. Jejunum segments were placed in baths containing Tyrode solution at 37°C, gassed with O2/CO2 and connected to an isotonic force transducer. Atropine (ATR) and tetrodotoxin (TDX) inhibited almost totally the spontaneous activity amplitude. Despite the presence of ATR or TDX, tissue contraction gradually recovered (within 5-10 min) to about 50% of baseline value; a second addition of ATR or TDX left the amplitude of the recovered contractions unchanged. Yet, after washout and a 45-min rest the contraction amplitude returned to baseline values and a further exposure to ATR or TDX markedly reduced it. In preparations pre-stimulated for 10 min with ACh, ATR abolished the TDX-resistant recovered spontaneous activity. Adrenaline and phenylephrine caused inhibition of tissue motility both in naïve and ATR- (or TDX)-exposed tissues; washout caused a rebound increase in contraction amplitude. Isoproterenol (up to 2.8 x 10-7 M) produced no inhibitory response in naïve tissues, but it caused (at 7.0 x 10-8 M) inhibition of the recovered spontaneous activity in tissues exposed to ATR or TDX, which was not affected by apamin. In naïve tissues, apamin caused a rapid and persistent increase in the contraction amplitude and blocked the inhibition by adrenaline and phenylephrine. The apamin-induced amplitude increase correlated with the rebound responses to adrenaline or phenylephrine. These results indicate that spontaneous motility in rabbit jejunum is predominantly mediated by neuronal release of ACh and by some other unidentified neuronal activity. Released ACh inhibits myogenic activity and strongly antagonizes b-adrenoceptor-induced apamin-insensitive inhibition but leaves a1agonist-induced apamin-sensitive inhibition unchanged.

Dept. of Pharmacology of Natural Substances and General Physiology, University of Rome "La Sapienza", Rome, Italy

P01-15

BRADYKININ INDUCES CHANGES IN [Ca2+]i IN EPITHELIAL NORMAL BREAST CELLS IN PRIMARY CULTURE

Greco S., Muscella A., Elia M.G., Cimaglia F., Storelli C., Marsigliante S.

The effects of bradykinin (BK) on intracellular calcium concentration ([Ca2+]i) in epithelial normal breast cells in primary culture were evaluated by using Fura 2-loaded cells. BK induced an increase of [Ca2+]i in a dose-dependent manner, showing maximal effect at 1 mM. 1 mM BK induced [Ca2+]i increase, after a 10-15 sec delay, to a peak of 678±45 nM above resting level (96±11 nM), and a subsequent decay to 165±37 nM. Both preincubation with B2 BK receptor and phospholipase C inhibitors blunted the BK effect, while pre-treatment with B1 BK receptor inhibitor did not, showing that B2 receptor and phospholipid hydrolysis are involved in BK signalling. The source of [Ca2+]i increase evoked by BK may be due to two mechanisms: release of Ca2+ by the intracellular Ca2+ pools and/or entry of Ca2+ through membrane channels. In this view, we incubated breast cells in Ca2+-free Krebs Ringer Hepes medium (KRH), without CaCl2 before BK stimulation; we found that the [Ca2+]i increase was reduced by 42% respect to the control, cells incubated in KRH with Ca2+, indicating that Ca2+ could entry through membrane channels. In addition, when 1.0 mM thapsigargin (TG), the inhibitor of the endoplasmic reticulum Ca2+ pumps, was used to discharge Ca2+ before BK treatment, BK increased the [Ca2+]i of only 50% with respect to the control, i.e. cells stimulated with BK only. This result indicates that the release of Ca2+ from TG-sensitive intracellular stores is involved in the BK-induced [Ca2+]i increase. The addition of 2 mM CaCl2 to cells previously treated with BK for 3.5 min in Ca2+-free KRH medium, induced a Ca2+ entry with a net peak height of 567±28 nM, indicating that stores operated membrane Ca2+ channels (SOCs) are involved in [Ca2+]i increase. In conclusion, in this study we demonstrated that in normal breast, BK through a functional B2 receptor evokes changes in [Ca2+]i by opening Ca2+ membrane channels and emptying intracellular stores.

Laboratorio di Fisiologia Generale-Dipartimento di Scienze e Tecnologie Biologiche e Ambientali – Lecce, ITALY

P01-16

MITOCHONDRIAL CONTROL OF CALCIUM SIGNALLING IN MOUSE EGGS AND ZYGOTES.

Dumollard RPL., Duchen M., Carroll J.

Similar to numerous somatic cells that elicit calcium oscillations upon stimulation, mammalian eggs respond to sperm entry by long lasting calcium oscillations. These calcium signals up-regulate mitochondrial ATP production by stimulating oxygen consumption and increasing the mitochondrial NADH concentration. However, in eggs, the effect of calcium signals on mitochondrial physiology remains unknown.

We imaged NADH and flavoprotein autofluorescence of mouse eggs, the mitochondrial electrical potential, simultaneously with the cytosolic Ca2+ concentration ([Ca2+]c). We manipulated mitochondrial oxidative phosphorylation by adding different substrates (Me-succinate, lactate, pyruvate, glucose) as well as perfusing inhibitors of the mitochondrial respiratory chain (Complexes I to V) onto mouse oocytes. Intracellular Ca2+ was manipulated by the addition of sperm or uncaging IP3. This experimental model is aimed at assessing potential roles for mitochondria in the regulation of the Ca2+ homeostasis in the oocyte and early embryo.

Perfusion of substrates and mitochondrial inhibitors perfusions revealed that mitochondria in eggs use the complex I of the respiratory chain (NADH-Ubiquinone oxidoreductase) to build up the electrical potential and synthesise ATP. Such synthesis of ATP by the mitochondria is necessary to maintain a low resting [Ca2+]c and to allow sperm-triggered Ca2+ oscillations. Confocal imaging of live oocytes showed that the mature mouse egg possesses numerous aggregates of phosphorylating mitochondria often embedded in sheets of endoplasmic reticulum. Finally we observed oscillations of the redox state (albeit without any mitochondrial potential changes) that are dependant on sperm-triggered Ca2+ oscillations or an IP3-mediated Ca2+ signal.

Together our observations provide evidence that, functional interactions exist between ER and mitochondria to regulate the pattern of calcium oscillation seen at the onset of development of the mouse embryo.

University College London, London, UNITED KINGDOM

P01-17

THE N-TERMINAL DOMAIN OF THE INOSITOL TRISPHOSPHATE RECEPTOR IS A TARGET FOR THIMEROSAL

Szlufcik K., Bultynck G., Nadif Kasri N., Callewaert G., Missiaen L., Parys J.B., De Smedt H.

The N-terminal domain (aa 1-225) of the type 1 inositol 1,4,5-trisphosphate (IP3) receptor (IP3R1) is considered as a suppressor of IP3 binding. In order to study the function of this domain we constructed a deletion mutant of mouse IP3R1 lacking those first 225 amino acids (D1-225) and expressed it in IP3R-knockout R23-11 B-lymphocytes. Although D1-225 was still able to bind IP3, it did not exhibit any measurable Ca2+-release activity. The thiol-reactive agent thimerosal potentiated the IP3-induced Ca2+ release and IP3-binding activity of the wild type mouse IP3R1 expressed in the R23-11 cells, but the stimulation of the binding could not be detected in cells expressing D1-225, suggesting that critical cysteine residues are lacking. By a 45Ca2+ flux technique a bell-shape dependence of the IP3 induced Ca2+-release on thimerosal is found, which was shifted to higher sensitivity in the presence of Ca2+. Using GST-IP3 binding core (aa 226-604) affinity chromatography, we identified an interaction between aa 1-225 and aa 226-604 of the mouse IP3R1. This interaction was regulated by Ca2+, strengthened by the addition of thimerosal and in the presence of this agent weakened by calmodulin and calcium-binding protein, whose binding sites are localized in the 1-225 region. The stimulatory effect of thimerosal for this interaction was mimicked by site-directed mutation of two well-conserved cysteine residues (C56A and C61A). Furthermore, GST-pull down experiments demonstrated a Ca2+-dependent interaction between aa 1-225, aa 226-604 or aa 1-604 and the C-terminus. These data provide evidence that amino acids residues 1-225 play an important role in the transduction of the activating stimulus from the IP3-binding domain to the gate of the channel. The target sites of thimerosal are localized presumably in this domain with cysteine residues C56 and C61 as possible candidates.

Laboratory of Physiology, Campus Gasthuisberg O/N, B-3000 Leuven, Belgium

P01-18

REGULATION OF INOSITOL TRISPHOSPHATE RECEPTORS BY PROTEIN-PROTEIN INTERACTIONS AND PHOSPHORYLATION

Vermassen E., Venmans E., Fissore R. A., Himpens B., Michalak M., Callewaert G., Missiaen L., De Smedt H., Parys J. B.

Most cell types express more than one type of inositol trisphosphate receptors (IP3Rs). Interestingly, these various IP3R isoforms can have different intracellular localizations. Moreover, their exact localization was recently shown to be dependent on the physiological state of the cell. The aim of this study was therefore to compare IP3R distribution in different cell types and to ascertain the mechanisms physiologically relevant for determining their localization. For this purpose, immunolocalizations experiments were supplemented by immunoprecipitation and phosphorylation analysis. In A7r5 smooth muscle cells, IP3R1 redistributed in a protein kinase C (PKC)-dependent and microtubule-dependent way by a mechanism most likely involving vesicle trafficking. IP3R3 however did not seem to redistribute. We therefore investigated IP3R localization in a number of cell lines that have IP3R3 as predominant isoform. In HeLa cells and K41 fibroblasts, both IP3R1 and IP3R3 display a homogenous distribution. In calreticulin-deficient K42 fibroblasts, no difference in subcellular localization of IP3R1 and IP3R3 was observed compared to the wild type. In bronchial epithelial cells (16HBE14o-), IP3R3 clusters were observed in the perinuclear region. Immunoprecipitation experiments in A7r5 cells, which contain both IP3R1 and IP3R3, demonstrated no interaction between either IP3R isoform with cytoskeletal proteins such as zyxin, vinculin or tubulin, while talin immunoprecipitated with both IP3R1 and IP3R3. To further investigate the role of PKC in the redistribution process, we investigated in which conditions and to what degree IP3R1 and IP3R3 could be phosphorylated by PKC. Purified IP3R1 and IP3R3 were both phosphorylated in vitro by PKC. These results indicate that in various cell types, IP3R isoforms are regulated by protein-protein interactions and by phosphorylation, which may be determinants of their intracellular localizations and function.

Laboratory of Physiology, Campus Gasthuisberg O/N, K.U.Leuven, B-3000 Leuven (Belgium)

P01-19

STIMULATION OF P2Y2 RECEPTOR INDUCES PROLONGED ACTIVATION OF ERK BY PKC-EPSILON.

Elia M.G., Greco S., Muscella A., Storelli C., Marsigliante S.

Extracellular purine nucleotides elicit a diverse range of biological responses through binding to specific cell surface receptors. Recently, we showed that in PC-Cl3 cells, a rat thyroid cell line that retains most of the features of differentiated follicular thyroid cells, ATP and UTP elevated the [Ca2+]i through the Gaq-coupled P2Y2 receptor.

To further elucidate the intracellular signalling mechanisms, we examined the effects of UTP on mitogen-activated protein kinase MAPK and proliferation. By Western blot analysis with an anti-phospho-p42/p44 MAPK antibody, we demonstrated that UTP activates ERK1/ERK2 in a time- and dose-dependent manner. The phosphorylation reached maximal levels after 3 min and returned to baseline in 6 h. ATP-induced activation of ERK1/ERK2 is dependent on the dual-specificity kinase mitogen-activated protein kinase/ERK kinase (MEK). In addition, UTP-stimulated MAPK activation was blocked by the protein kinase C (PKC) inhibitors staurosporine but not by Gö 6976, a preferential inhibitor of calcium-dependent PKC isoforms. The involvement of PKC in the signal transduction pathways was further supported by the ability of UTP to induce translocation of PKC-epsilon. PKC- epsilon isoform was translocated by a 0.5 min UTP stimulation and returned to the cytosol after 15 min. In many cells, the extracellular signal-regulated kinase (ERK) cascade plays an important role in cellular proliferation. We evaluated the effects of UTP on PC-Cl3 cell proliferation by a) a spectrophotometric 3-(4,5-dimethylthazol-2-yl)–2,5-diphenyl-2H tetrasodium bromide (MTT) assay; b) direct cell count and c) total protein assay. PC-Cl3 cells were incubated with different concentrations of UTP (0.1, 1 and 10) for 24 and 48 hours. UTP had no effects on cellular proliferation and total cellular protein. In conclusion, UTP induced prolonged activation of ERK1/2 through PKC- epsilon without affecting cell proliferation.

Lab. di Fisiologia Gen.-Dip. di Scienze e Tecn. Biol. e Ambientali-Università di Lecce - ITALY

P01-20

ENDOPLASMIC RETICULUM MORPHOLOGY AND POLARITY OF Ca2+ SIGNALLING IN PANCREATIC ACINAR CELLS

Gerasimenko O.V., Gerasimenko J.V., Rizzuto R. R., Treiman M., Tepikin A. V., Petersen O. H.

Pancreatic acinar cells are highly polarised cells with a distinct secretory granule area; the mitochondria are positioned outside the secretory granule region. The basal part of the cell contains the nucleus and the highly developed endoplasmic reticulum. It has been shown that IP3-induced calcium release initiates in the secretory granule area, which contains very little ER. Using confocal and two-photon microscopy, we investigated the morphology of the ER in the secretory granule area of living pancreatic acinar cells. The positioning of the ER was compared with the distribution of other cellular organelles. We found, that although the main part of the ER is located in the basal part of the cells, there are strands of the ER in the secretory granule area. The strands of ER projecting into the granular region are connected with the main ER structures in the basal area of the cells. This is the first visualization of the ER strands in the secretory granule area in living pancreatic acinar cells. The density of the ER decreases abruptly at the apical/basal border. These data confirm our recent findings demonstrating the tunnel function of the ER, which allows high Ca2+ mobility in the ER lumen. Ca2+ is released from the ER terminals in the granular area and this Ca2+ releasable store is re-supplied from the main calcium store at the basal area of the cell by the ER tunnel function.

Physiology Dept, Liverpool Univ, Liverpool, UNITED KINGDOM

P01-21

MEMBRANE IP3 RECEPTOR IS MEDIATOR OF THE SYNERGISM BETWEEN ATP AND ADENOSINE ON THE CILIARY BEAT

Barrera N., Torres S., Morales B., Villalon M.

ATP and adenosine induce a synergistic increase of the ciliary beat frequency (CBF) in cultured ciliated cells from hamster oviduct. To elucidate the mechanism involved in this interaction, we quantified the intermediaries of the ATP transduction pathways in the presence of adenosine. ATP activates the phospholipase C followed by IP3 receptor activation. Using the immunogold and electronic microscopy, the subcellular distribution of IP3 receptors types 1 and 3 in oviductal ciliated cells determined the presence of both receptors types in nucleus and reticulum endoplasmic, however only the type 3 was localized in plasma membrane. Using fluorescence spectroscopy, it was demonstrated that ATP or caged IP3 increased the intracellular Ca2+ free concentration ([Ca2+]i), initially from intracellular reservoirs followed by a Ca2+ influx. Addition of adenosine or intermediaries of adenosine transduction pathways, such as 8 Br-cAMP (a cAMP permeable analogue) or protein kinase A (PKA) in the presence of ATP, induced a higher Ca2+ influx. Furthermore Ca2+ influx induced by caged IP3 was increased by the release of caged cAMP. Using the radioimmumoreceptor technique, it was observed a high correlation between the time course of the IP3 production and both sources of the [Ca2+]i increase. Using the patch clamp technique in whole cell recording, ATP triggered an entry of Ca2+ which is blocked by Xestospongin C, a IP3 receptor inhibitor. In the presence of adenosine, we observed a higher ATP dependent-Ca2+ current, which is diminished by H-89, a PKA blocker. Furthermore, in the inside-out configuration, IP3 and the catalytic PKA subunit triggered a higher Ca2+ current compare to IP3 alone. These results suggest that the synergism of CBF increase between ATP and adenosine depend on IP3 receptor membrane activation by ATP and the modulation of these receptors by PKA dependent adenosine activation. Supported by CONICYT and FONDECYT 2010120.

Pontificia Universidad Catolica de Chile, Santiago, Chile. Universidad de Valparaiso. Universidad de Santiago de Chile.

P01-22

FUNCTION, BUT NOT LOCATION OF MITOCHONDRIA, IS CRITICAL TO SUSTAIN STORE-OPERATED Ca2+ INFLUX

Frieden M., Castelbou C., James D., Martinou J.C., Demaurex N.

Mitochondria modulate, propagate, and synchronize Ca2+ signals by taking up and releasing Ca2+ at key locations near Ca2+ release or influx channels. Functional mitochondria are required to sustain the activity of store-operated Ca2+ channels (SOC) at the plasma membrane, but it is not clear whether mitochondria act as local Ca2+ buffers to remove Ca2+-dependent channel inhibition or release a diffusible messenger. The location of mitochondria relative to SOC channels is difficult to ascertain, as mitochondria are dynamic structures that form a tubular network constantly remodeled by fusion and fission reactions. To distinguish between local and global effects of mitochondria on SOC channels, we transiently transfected HeLa cells with hFis1, a protein that promotes mitochondria fission. hFis1 expression induced mitochondrial fragmentation within 4h, all mitochondria appearing as punctuate organelles clustered around the nucleus. Despite the dramatic morphological change, the mitochondrial membrane potential and pH as well as the amplitude of mitochondrial Ca2+ transients, measured with targeted ratiometric pericam, were not altered by hFis1 expression. However, upon Ca2+ readdition to histamine-stimulated cells hFis1-fragmented mitochondria took up Ca2+ with a significant delay, consistent with their increased distance from the cell membrane. The delayed transfer of Ca2+ was not due to reduced Ca2+ entry, as hFis1 did not affect the amplitude and kinetics of cytosolic Ca2+ changes upon Ca2+ readdition. Regardless of hFis1 expression and mitochondria location, disruption of mitochondrial potential with oligomycin/rotenone or CCCP reduced Ca2+ entry by ~40%. These observations indicate that mitochondria remain functional despite drastic alteration in their morphology. Sustained Ca2+ entry requires functional mitochondria but not the presence of mitochondria near membrane channels, indicating that mitochondria exert a global, rather than a local effect on SOC channels.

University of Geneva, Dpt of Physiology, Medical Center, Geneva, Switzerland.



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