Federation of European Physiological Societies
3rd FEPS Meeting
Nice, 28 June – 2 July 2003
Société de Physiologie
The edition of the abstract book has been supported by
The Société de Physiologie
Table of Contents
S1 : Calcium signalling 8
ORAL SESSION 8
POSTER SESSION 10
S2 New aspects of ionic transport (I) The Pflugers Archiv Symposium 17
ORAL SESSION 17
POSTER SESSION 19
S3 Environmental physiology 25
ORAL SESSION 25
POSTER SESSION 27
S4 Blood pressure regulation 36
ORAL SESSION 36
POSTER SESSION 38
S6 Sensors and effectors in body fluid homeostasis 46
ORAL SESSION 46
POSTER SESSION 48
S7 Cellular and Molecular aspect of renal physiology 51
ORAL SESSION 51
POSTER SESSION 53
S8 Control of calcium transport in the heart : physiology and pathophysiology 59
ORAL SESSION 59
POSTER SESSION 61
S9 Extracellular (Volume) transmission : its mechanisms and function 65
ORAL SESSION 65
POSTER SESSION 67
S12 Responses to osmotic challenges 69
ORAL SESSION 69
POSTER SESSION 71
S27 Cell Volume: Regulation and Functional Impact 73
ORAL SESSION 73
POSTER SESSION 75
S13 New aspects of ionic transport (II) 78
ORAL SESSION 78
POSTER SESSION 80
S14 Calcium signalling and neuronal glial interactions 85
ORAL SESSION 85
POSTER SESSION 87
S15 The new integrative brain physiology 88
ORAL SESSION 88
POSTER SESSION 90
S16 Role of Gap junctions in vascular tissue 100
ORAL SESSION 100
POSTER SESSION 102
S17 Aldosterone: synthesis, targets and functions 104
ORAL SESSION 104
POSTER SESSION 106
S18 Physiology of eating behaviour and energy expenditure: control by central and peripheral mechanisms 108
ORAL SESSION 108
POSTER SESSION 110
S19 Physiology of reproduction 114
ORAL SESSION 114
POSTER SESSION 116
S20 Dynamic analysis of biological oscillators 120
ORAL SESSION 120
POSTER SESSION 122
S21 From gene to muscle function 124
ORAL SESSION 124
POSTER SESSION 126
S22 Oxygen Signalling 133
ORAL SESSION 133
POSTER SESSION 135
S23 K+ Channels 141
ORAL SESSION 141
POSTER SESSION 143
S24 Pulmonary circulation : from cell to integrative physiology 149
ORAL SESSION 149
POSTER SESSION 151
SPATIAL AND TEMPORAL ASPECTS OF CALCIUM SIGNALLING
Michael J. BERRIDGE
Calcium (Ca2+) is a highly versatile intracellular signal capable of regulating many different processes. To achieve this versatility, the signalling system operates in many different modes thus enabling it to function over a wide dynamic range. At the synaptic junction, for example, Ca2+ triggers exocytosis within microseconds, whereas at the other end of the scale Ca2+ has to operate over minutes to hours to drive processes such as gene transcription and cell proliferation. At any moment in time, the level of intracellular Ca2+ is determined by a balance between the ON reactions that introduce Ca2+ into the cytoplasm and the OFF reactions during which this signal is removed through the combined action of buffers, pumps and exchangers.
Cells have access to a very extensive Ca2+ signalling toolkit from which each cell type expresses a unique set of components to create Ca2+ signalling systems with widely different spatial and temporal properties. Spatial properties are particularly relevant for the fast responses where components of the ON reactions and their downstream effectors are closely associated. This spatial contiguity is less apparent for the slower responses such as gene transcription, fertilization and cell proliferation where Ca2+ signals tend to operate more globally and where temporal properties of signalling become increasingly important in that signalling is usually presented in the form of repetitive Ca2+ transients and waves. The Ca2+-sensitive processes have effector systems tuned to respond to particular Ca2+ transients. At the fast end of the scale, such as synaptic transmission or cardiac contraction, the effector systems respond to pulses within the micro- to millisecond range. As one moves up the time scale, the transients tend to last for longer (seconds to minutes) and the resulting signal spreads out as a Ca2+ wave to reach targets distributed throughout the cell. During prolonged stimulation, these transients are repeated to set up regular Ca2+ oscillations that have been implicated in the control of many different processes.
Many of these Ca2+ signalling systems are organized into macromolecular complexes enabling Ca2+ to carry out its signalling function within a highly localized environment. These complexes can function as autonomous units or modules that can be multiplied up or mixed and matched to create larger more diverse signalling systems. A typical example is the cardiac Ca2+ release unit that can be recruited independently of its neighbours to produce graded contractions. This highly organized cardiac Ca2+ signalling unit illustrates a number of the important dynamic aspects of the ON/OFF reactions of Ca2+ signalling such as amplification, homeostasis, tunnelling and modulation through cross talk with other signalling pathways.
Such Ca2+ signalling systems are not fixed in stone, but are constantly being remodelled to adapt to changing circumstances to ensure that each specific cell type continues to deliver the Ca2+ signals that characterizes its unique function. If the spatiotemporal properties of this output signal change due to a loss or defect of a key component, compensatory mechanisms come into play to restore the normal output signal. This remodelling process implies an element of quality assessment in that the output of the signalling system is under constant review. It seems that Ca2+ itself plays a critical role in this internal assessment mechanism by remodelling its own signalling pathway.
Evidence to support this hypothesis of Ca2+-induced Ca2+ signalling remodelling is the fact that Ca2+ is a potent activator of gene transcription and that some of these genes are known to code for components of the Ca2+ signalling toolkit such as the expression level of Ca2+ signalling components such as pumps and channels. For example, expression of the InsP3 receptor is mediated through the calcineurin/NFAT transcriptional cascade.
A number of important disease states (hypertension, congestive heart failure, manic depressive illness, Alzheimer’s disease) may result from abnormal remodelling of Ca2+ signalling systems. A good example is congestive heart failure, a major cause of human morbidity and mortality, which usually develops when the heart tries to adapt to stress usually in the form of an increased workload. The initial response is for the heart to grow and to begin to display an altered phenotype by expressing neonatal genes. This hypertrophy is a compensatory mechanism in that the heart will return to its original phenotype and size if the abnormal inputs are reduced. However, if the stresses persist, this compensated hypertrophy shifts to the more irreversible state of congestive heart failure. The phenotypic remodelling that occurs during both cardiac hypertrophy and congestive heart failure is controlled by a number of signalling pathways of which Ca2+ seems to play a prominent role.
A major problem with trying to understand how Ca2+ controls cardiac hypertrophy is the fact that the heart is not quiescent but is continuously subjected to large periodic Ca2+ signals that flood through the cytoplasm and nucleus every time the heart contracts. Why is it then that cardiac cells that are subjected to this constant barrage of Ca2+ avoid triggering a hypertrophic response? It has been suggested that the normal functioning heart may not be transcriptionally silent but may be under constant Ca2+-dependent surveillance. If this is the case, then the increase in transcription during hypertrophy may result from subtle differences in the spatiotemporal properties of the individual Ca2+ spikes. Indeed, a broadening of the Ca2+ transient or an increase in its amplitude have been recorded in cases where hypertrophy is induced by modifying the levels of proteins such as triadin or FKBP12.6. It is a change in the kinetics of the Ca2+ transient that seems to carry the information responsible for inducing hypertrophy. Such subtle changes in Ca2+ signalling might be sufficient to activate a distinctive programme of gene transcription.
A different phenotypic remodelling process, which appears to be irreversible, occurs during the onset of congestive heart failure when the Ca2+ signalling system is severely down regulated. The most noticeable change is a dramatic decline in the activity of the SERCA pump caused by a decrease in its mRNA and protein expression level. This decline in SERCA2 activity coincides with an increase in the activity of the NCX1, which will reduce the access of the SERCA2 pump to Ca2+ thus contributing to the severe depletion of the SR store that characterizes congestive heart disease.
Laboratory of Molecular Signalling, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK.
ION CHANNELS AND ASSOCIATED PATHOLOGIES
Ion channels are the targets of numerous drugs with antihypertensive, antidiabetic, antiepileptic or analgesic effects. The presentation will deal with the analysis of the molecular properties, pharmacology and relation with disease states of two new families of ion channels.
The first one consists of the 2P-domain K+ channels. This class of channels plays a central role in the regulation of resting potentials as well as in the shaping of action potentials. Two particular sub-families will be discussed. The first one is that of TREK and TRAAK channels. These channels are mechanosensitive, but they are also activated potently by polyunsaturated fatty acids and lysophospholipids as well as by intracellular acidification (TREK). The second one comprises TASK channels that are involved in sensing small extracellular pH variations. Both TREK and TASK channels are regulated by numerous neurotransmitters. Through their action on both TREK and TASK channels, metabotropic receptors have a potent ionotropic function. Both TREK and TASK channels are major targets in the action of volatile anaesthetics. TREK and TRAAK channels are essential targets in neuroprotection against brain and spinal cord ischemia as well as against epilepsy and spectacular effects can be obtained by activating them. TREK channels also have a sensory function in nociceptors and may be involved in psychiatric diseases.
The second new family of ion channels (ASICs) is permeable to Na+. The ASIC channels are non-voltage activated, proton-activated ion channels. They are the most simple ligand gated channels and are involved in sensing extracellular acidifications including those that probably occur at post-synaptic levels. They are present everywhere in brain as well as in nociceptors and they seem to be particularly important in pain perception. Their molecular properties as well as their pharmacology and their involvement in nociception will be discussed.
Institut de Pharmacologie Moléculaire et Cellulaire – CNRS UMR 6097 – 660, route des Lucioles, Sophia-Antipolis, 06560 Valbonne - France
NEW ASPECTS OF RENAL POTASSIUM TRANSPORT
Gerhard GIEBISCH, Steven HEBERT and Wenhui WANG
The kidney’s major role in potassium (K) homeostasis depends on its ability to respond effectively to changes in external K balance and to stabilize the extracellular concentration of K. The correction of deviations from normal plasma K levels and the maintenance of external K balance depend on the intrinsic ability of distal nephron segments to either secrete or reabsorb K. Following extensive reabsorption of K along the proximal tubule and the thick ascending limb of Henle’s loop, net K secretion occurs mainly in principal cells. K secretion is suppressed in K depletion and replaced by K reabsorption in intercalated cells. Studies on single tubules and principal and intercalated cells have defined the determinants of K secretion and reabsorption including the electrochemical driving forces, specific carriers, ATPases and K channels. Recent studies on the properties and molecular identity of renal K channels have also contributed significantly to understanding the renal mechanisms that transport and regulate K excretion.
Department of Cellular and Molecular Physiology, Yale University, New Haven, CT and Department of Pharmacology, New York Medical College, Valhalla, NY
ATP-SENSITIVE K CHANNELS AND INSULIN SECRETIONIN HEALTH AND DISEASE
University Laboratory of Physiology - Parks Road, Oxford, UNITED KINGDOM
(abstract not received)
FUNCTIONAL ARCHITECTURE OF THE NICOTINIC RECEPTOR AT THE AMINO ACID LEVEL : A MEMBRANE ALLOSTERIC PROTEIN
The nicotinic receptor is a transmembrane hetero-pentamer of 300MW which carries the binding sites for acetylcholine, and the ion channel together with structural elements which account for its fast opening and slow desensitization by the neurotransmitter (Changeux & Edelstein, 1998 ; Karlin, 2002 ; Unwin, 1999). The amino acids which compose the ACh binding sites have been identified by photolabeling and by site-directed mutagenesis. They belong to 6-distinct loops within the large NH3-terminal hydrophilic domain of the receptorsubunits located at the interface between subunits and including an a-subunit (Corringer et al., 2000). Molecular modeling of the ACh binding pocket on the basis of the Xray structure of snail acetylcholine binding protein offers opportunities for docking and thus for design of nicotinic ligands at various neuronal subunit interfaces (Le Novère et al., 2002) Furthermore, in T. marmorata, the relative contribution of several different loops changes upon stabilization of the high-affinity desensitized state by the allosteric effector meproadifen (Galzi et al., 1991). The channel blocker chlorpromazine covalently labels, upon UV irradiation, all the subunits when bound to its unique high-affinity site located in the ion channel. The labeled amino acids belong to the hydrophobic segment MII and form three superimposed rings assuming MII helical (Giraudat et al., 1986, 1987 ; Hucho et al., 1986 ; Revah et al., 1991). Mutations, within or in the neighborhood, of MII from 7 neuronal nicotinic receptor selectively alter the ionic selectivity for Ca++ vs Na+/K+ (Bertrand et al., 1993) and for cations vs anions (Galzi et al., 1992). On the basis of systematic mutagenesis experiments, a model of the ion channel is proposed which locates the ion selectivity filter at the level of the cytoplasmic loop linking MII and MI (Corringer et al., 2000). Mutations of AA rings from MII cause "gain of function" pleiotropic phenotypes with, altogether, loss of desensitization, enhanced affinity for agonists and conversion of the competitive antagonists into agonists (Révah et al., 1991 ; Bertrand et al., 1992). Homologous genotypes and phenotypes have been recently identified in human patients with congenital myasthenia gravis and frontal lobe nocturnal epilepsy (rev. Ohno & Engel, 2002 ; Raggenbass & Bertrand, 2002). The data are interpreted in terms of a four-state "allosteric" model (Edelstein et al., 1997 ; Changeux & Edelstein, 1998 ; Corringer et al., 2000). Furthermore, obtention of functional chimaera between 7 and 5HT3 receptors reveals a functional autonomy of the neurotransmitter binding and channel domains (Eiselé et al., 1993) supporting a common functional organization of these different receptors (Le Novère et al., 1999, 2002).
Implications of the allosteric model to the understanding of short-term memory and cognitive functions are discussed (Heidmann & Changeux, 1982 ; Dehaene & Changeux, 1997 ; Dehaene et al., 1998).
CNRS URA 2182 "Récepteurs et Cognition", Institut Pasteur,
25 rue du Dr Roux, 75015 Paris, France.
THE REGULATION OF EPITHELIAL Na+ CHANNELS IN EXOCRINE EPITHELIA
David I. COOK
Epithelial Na+ channels are expressed in the apical membranes of epithelia such as the renal collecting duct, the distal colon, the respiratory epithelium and the ducts of salivary and sweat glands. They are a key component in the mechanism by which these epithelia transport Na+ ions across their apical membranes and have been shown to regulate blood pressure and extracellular fluid volume. They also play a critical role in controlling the thickness of the fluid layer covering the surface of the respiratory and gastrointestinal tracts. Abnormalities in the structure and regulation of these channels have been implicated in the pathogenesis of the autosomal dominant form of hypertension, Liddle’s syndrome, salt-wasting conditions such as pseudohypoaldosteronism type I and cystic fibrosis. Consistent with the critical role of epithelial Na+ channels in the normal regulation of blood pressure, of extracellular fluid volume and of the thickness of the fluid on the respiratory surfaces, transgenic mice in which the a-subunit of the channel has been deleted by homologous recombination die at birth due to failure to clear their lungs of fluid, and transgenic mice in which expression of the b- or g-subunits has been reduced, suffer from a salt-losing nephropathy characterised by hyperkalaemia and hypotension.
Given their importance for the regulation of the milieu intérieur, it is not surprising that epithelial Na+ channels are regulated by a wide variety of hormones, including aldosterone, ADH and insulin-like growth factor I. They are also regulated by feedback systems that adjust the activity of the channels to ensure that the rate of Na+ entry to the cytosol across the apical membrane does not exceed the capacity of the Na+-K+-ATPase to extrude it across the basolateral membrane. These feedback systems thus operate to ensure stability of the volume and ionic composition of the cytosol.
The nature of these feedback systems has been the subject of investigation since their existence was first postulated by Ussing and MacRobbie over 40 years ago. The mechanisms that have been proposed include: (i) inhibition of the channels by an extracellular modifier site that binds extracellular Na+, (ii) direct inhibition of the channels by increased intracellular Na+, (iii) inhibition of the channels by increased intracellular Cl- serving as a surrogate for cell volume, (iv) inhibition of the channels by the increase in intracellular Ca2+ which results from the slowing of Na+-Ca2+ exchange produced by increases in intracellular Na+, and (v) inhibition of the channels by the decrease in cytosolic pH which results from the slowing of the Na+-H+ exchanger produced by increased intracellular Na+. Of these, the Na+ and the Cl-feedback systems are of particular interest.
The Na+ feedback system is mediated by an ubiquitin-protein ligase, either Nedd4 or Nedd4-2, which binds to proline rich domains, the so-called PY motifs, in the b- and g-subunits of the Na+ channels. Once bound to the channels, the ubiquitin protein ligases ubiquitinate and inactivate them. In mouse mandibular duct cells, it has been possible to further show that the concentration of intracellular Na+ is sensed by a cytosolic receptor that can be blocked by compounds such as benzimidazolylguanidinium (BIG) and dimethylamiloride (DMA) which have been reported to block Na+ feedback regulation in intact tissues. This receptor in turn activates the G protein, Go, the a-subunit of which then activates the ubiquitin protein ligase. Many cases of Liddle’s syndrome are due to the mutation or deletion of the PY motif in the b- or the g-subunit of the Na+ channel, the increased channel activity seen in this condition being attributable to disruption of the Na+ feedback system. There have also been recent reports that the hormonal regulators of epithelial Na+ channel activity, aldosterone and IGF-I, activate epithelial Na+ channels as a consequence of increasing the activity of the serum- and glucocorticoid-inducible kinase, Sgk, which in turn phosphorylates the ubiquitin-protein ligase, Nedd4-2, leading to interruption of the Na+ feedback regulatory system. Given that epithelial Na+ channels are known to be extensively phosphorylated in vivo, these reports raise the issue of the extent to which other kinases may be able to modulate the activity of the Na+ feedback system.
The Cl- feedback system, on the other hand, does not involve ubiquitin-protein ligases or ubiquitination of the channel. In mouse mandibular duct cells, it has been shown to be mediated by the G protein, Gi2. Like the Na+ feedback system, the Cl- feedback system is probably mediated by an intracellular receptor for Cl, although it has not yet proven possible to demonstrate this conclusively. The Cl- feedback system is not sensitive to agents such as BIG, although it is blocked, as is the Na+ feedback system, by extracellular exposure of the channels to sulfyhydryl reactive reagents such a r-chloromercuriphenylsulfonate. Recently, Cl- feedback regulation has been proposed as the mediator of the inhibitory action of the Cl- channel, CFTR, on epithelial Na+ channels. If this is correct, then the aberrant regulation of cytsolic Cl-which accompanies mutations of CFTR would be the cause of the increased epithelial Na+ channel activity that is observed in cystic fibrosis.
As mentioned above, epithelial Na+ channels play a critical role in regulating the thickness of the fluid layer that coats the surface of the respiratory epithelium. Increased activity of the channels leads to dehydration of the surfaces of the respiratory epithelium as is observed, for example, in cystic fibrosis. Conversely, decreased activity of the channels, as is observed in the hereditary condition, pseudohypoaldosteronism type I, leads to fluid accumulation in the lungs and respiratory passages. Recently, it has become evident that many acquired diseases associated with the accumulation of fluid in the lungs or in other parts of the respiratory tract, such as high altitude pulmonary oedema, respiratory distress syndrome and otitis media, are associated with decreased activity of the Na+ channels in the lining epithelium. In particular, many pathogens, including both gram-negative bacteria and viruses, inactivate epithelial Na+ channels. In the case of influenza virus this inactivation is due to the hemagglutinin in the viral coat binding a glycoprotein in the apical membrane in the epithelial cells, which in turn activates protein kinase C leading to inhibition of channel activity. Other pathogens appear to act by triggering the release of ATP which then acts in an autocrine manner on purinergic receptors in the apical membrane to inhibit the Na+ channels. Irrespective of the detailed mechanism, the reduction in the rate of Na+ transport will shift the net rate of fluid transport by the epithelium towards secretion and promote such manifestations of respiratory infections as pulmonary oedema, sinusitis and rhinorhoea.
Department of Physiology F13, University of Sydney, NSW 2006, Australia
GENOMICS AND PHYSIOLOGY
INSERM Nantes – France
(abstract not received)
EVOLUTION OF THE GENETIC MAP OF CARDIOVASCULAR FUNCTION
Allen W. COWLEY, Jr
The genetics of multifactorial disorders such as hypertension, arthritis, and diabetes in human populations has proven to be very challenging due to the modest nature of gene effects and the heterogeneity of patient populations. With the genetic sequencing of the human, mouse, and rat genomes now nearly completed, there is a need to define and place gene function in the context of complex systems biology. This lecture will focus upon two experimental approaches that have begun to provide an understanding of the relationships among genes, environmental stressors, and blood pressure. The first utilizes linkage studies with total genome scans. This approach has led to the first genomic-systems biology map of cardiovascular function. In studies performed in the F2 offspring of an intercross between the Dahl salt-sensitive rat (SS./Mcw) and the Brown Norway rat (BN), more than 200 cardiovascular phenotypes were determined during normal and stressed conditions. Genomic regions accounting for a large degree of the variability of 81 of the traits in the male population and 126 in the female population were identified and mapped as quantitative trait loci (QTL) regions on the rat genome. In addition, a number of QTL of cardiovascular and renal traits were found to be determined by gender and mapped to discrete regions of the genome. The results of these linkage analyses have led to a richly annotated map of genome function. The second approach has been the development of consomic panels of inbred rats that are enabling a broad mapping of cardiovascular pathways and leading to more detailed identification of genes related to these pathways and providing controls for genetic background effects. BN chromosomes are introgressed onto the DS genomic background one chromosome at a time in each strain. Each inbred consomic rat strain enables the assessment of the contribution of genes specific to that chromosome and provides strains with uniform genetic backgrounds for various genetic and physiological studies. Environmental stressors such as hypoxia, exercise, and high salt intake are being used to unmask deficiencies in normal homeostatic mechanisms and idiopathic mechanisms that contribute to disease as determined by more than 300 measured phenotypes to characterize heart, lung, kidney, vasculature, and blood function. Comparative mapping strategies are used to link these traits to the genomes of the mouse and human. As major phenotypic differences are identified within the consomic stains, these strains are being made commercially available (Charles Rivers, Inc) and provide highly useful model systems to test both physiological and genetic hypotheses. When combined with the rapid derivation of even more discrete chromosomal substitutions of very narrow regions within chromosomes (congenic strains) together with DNA microtechnology and proteomics, these functionally and genomically annotated rat strains provide powerful new tools to link complex systems biology to related genetic pathways.
Dept. of Physiology, Medical College of Wisconsin, Milwaukee, WI., USA.
S1 : Calcium signalling
LOCALIZATION OF Ca2+ TRANSPORTERS IN EXOCRINE ACINAR CELLS
More than 30 years ago, I showed that neurotransmitters acting on exocrine gland cells release Ca2+ from a store in the endoplasmic reticulum (ER). 20 years ago, work on pancreatic acinar cells by Michael Berridge and his collaborators provided the original evidence for the Ca2+ releasing action of IP3. Ironically, further work on the pancreatic acinar cells presented some problems for the view that IP3 acts on the ER, since the primary Ca2+ release site was in the apical pole, which contains the secretory granules, but little ER. All cytosolic Ca2+ signal responses to stimulation with neurotransmitters, hormones and intracellular messengers (including IP3, cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate) are initiated in the secretory granule area and are mostly confined to this region. These local Ca2+ signals control not only exocytosis, but also fluid secretion via regulation of Ca2+-activated Cl-channels in the apical plasma membrane. We have mapped the Ca2+-sensitive Ca2+ release sites, using local uncaging of caged Ca2+, and shown that Ca2+-induced Ca2+ release (which does not involve IP3 formation) can only be triggered in the apical pole and is dependent on both functional IP3 and ryanodine receptors. Ryanodine itself triggers Ca2+ waves, which always start in the apical pole. The distribution of ER in living acinar cells, visualized by ER-specific fluorescent probes with confocal and two-photon microscopy, shows that although the bulk of the ER is located in the basolateral area, there is significant invasion of ER into the granular pole and each secretory granule is surrounded by ER strands. This provides the framework for a coherent and internally consistent theory for cytosolic Ca2+ signal generation in the secretory pole, where the primary Ca2+ release occurs from ER terminals supplied with Ca2+ from the main store at the base via the tunnel function of the ER.
Department of Physiology, University of Liverpool, UNITED KINGDOM
THE ROLE OF MITOCHONDRIA IN THE GENESIS OF THE CALCIUM SIGNAL IN EXCITABLE CELLS
Garcia-Sancho J., Alonso M.T., Villalobos C.
Ca2+ transport by organella contributes to shaping Ca2+ signals and exocytosis. Therefore, accurate measurements of [Ca2+] inside organella are essential for a comprehensive analysis of the Ca2+ redistribution that follows cell stimulation. On the other hand, [Ca2+] inside organella regulates by itself important physiological functions. Here we have combined virus-based expression of targeted aequorins with photon counting imaging to resolve dynamics of the cytosolic and mitochondrial Ca2+ signals at the single-cell level. Adrenal chromaffin and anterior pituitary cells were used as models for excitable cells.
On activation of plasma membrane voltage-gated Ca2+ channels, mitochondria took up large amounts of calcium through the mitochondrial Ca2+ uniporter. Results are consistent with the generation of cytosolic high-Ca2+ subplasmalemmal domains adequate for triggering exocytosis. At the cell core, a smaller increase of cytosolic Ca2+, adequate for recruitment of the reserve pool of secretory vesicles to the plasma membrane, is produced. Most of the entering Ca2+ load is taken up by a mitochondrial pool, M1, closer to the plasma membrane. The increase of mitochondrial[Ca2+] stimulates respiration in these mitochondria, thus providing local support for the exocytotic process.
Anterior pituitary cells exhibit spontaneous electric activity and cytosolic Ca2+ oscillations that are responsible for basal secretion of pituitary hormones and are modulated by hypophysiotrophic factors. Aequorin reported spontaneous [Ca2+] oscillations in bulk cytosol, nucleus and mitochondria. Interestingly, a fraction of mitochondria underwent much larger Ca2+ oscillations, which were driven by local cytosolic high-[Ca2+] domains generated by the spontaneous electric activity. These oscillations were large enough to stimulate respiration, providing the basis for local tune-up of mitochondrial function by the Ca2+ signal.
Instituto de Biologia y Genetica Molecular (IBGM), Univ. Valladolid & CSIC - Spain
THE CALCIUM SIGNALLING ACTIVATED BY NORADRENALINE IN RAT ARTERIES IS REGULATED BY RHO-KINASE
Morel N., Ghisdal P., Vandenberg G.
In vascular smooth muscle cells, contractile agonists activate a complex chain of events to increase cytosolic Ca concentration and contract the arteries. The present study was aimed at investigating the potential role of Rho-kinase in the Ca signal activated by noradrenaline in rat aorta and mesenteric artery.
In fura-2 loaded arteries, the Rho-kinase inhibitor Y-27632 (10 µM) completely relaxed the contraction evoked by noradrenaline (1 µM) and simultaneously inhibited the Ca signal by 54 ± 1 % (mesenteric artery) and 71 ± 15 % (aorta), while in KCl-contracted arteries, Y-27632 decreased tension without changing cytosolic Ca. Similar effects were obtained with another inhibitor of Rho-kinase (HA 1077), but not with an inhibitor of protein kinase C (Ro-31-8220). In aorta bathed in Ca-free solution, noradrenaline response consisted of a rapid but transient increase in cytosolic Ca. Re-addition of Ca into the Ca-free solution evoked a slow, sustained increase in Ca signal, which was partly inhibited by 10 µM Y-27632 (=65 %) and completely blocked by 1 µM Gd. In the presence of nimodipine, 10 µM Y-27632 or 1 µM Gd3+ completely blocked the entry of Ba activated by noradrenaline. However, Y-27632 did not affect the production of inositol phosphates activated by noradrenaline, and the release of Ca from the sarcoplasmic reticulum evoked by IP3, measured by the activation of Ca-dependent K current using the patch-clamp technique. Finally, Y-27632 did not inhibit the Ca signals evoked by thapsigargin or by caffeine and the capacitative Ca entry activated by the depletion of intracellular Ca stores by thapsigargin.
These results indicate that Rho-kinase does not alter the capacity of intracellular Ca storage and release, but that it is involved in the activation of a phospholipase C-dependent Ca entry pathway in rat arteries.
Université catholique de Louvain - Laboratoire de Pharmacologie – BELGIUM
AN INTERACTIVE COMPUTER PROCEDURE FOR AUTOMATIC DETECTION AND MEASUREMENT OF MUSCLE CALCIUM SPARKS
Sebille S., Cantereau A., Vandebrouck C., Balghi H., Constantin B., Raymond G., Cognard C.
In muscle cells, contraction is controlled by Ca2+ ions, which are rapidly released from the sarcoplasmic reticulum during sarcolemmal depolarization. In addition to this synchronised spatially homogeneous calcium signal, discrete calcium release events, termed sparks, have been discovered with the use of confocal microscopy in diverse tissues as skeletal, cardiac, and smooth muscle. Determination of the calcium spark morphology parameters is of critical importance to understand the nature of elementary calcium release events in muscle. Because of multiple behaviours of release, it appeared necessary to evaluate parameters from several hundreds of sparks in a same cell population in order to obtain reliable statistics. Automatic detection algorithms without user intervention have been previously developed on single images to automatically detect and analyse sparks in confocal line scan (space-time: 512 * 512 pixels) images. Nevertheless, most of previous studies have been performed on isolated sparks without taking into account that events could originate from the same locus of release. Our first studies on myotubes clearly showed that many events were originating from the same space location. Thus, we have addressed the problem of recognizing polymorphic events on series of images in order to follow sparks morphology from one site during several seconds. Here, we describe an interactive procedure coded in the image-processing language IDL 5.3. , that can be applied on series of n images (512 x 512 x n) derived from the same scanning line. Computing simultaneously entire series of images permits to measure, with the conventional morphological parameters, location and frequency of release from each release site. The use of this procedure provides quickly much information about the properties of release sites in muscle cells and can be applied on any elementary calcium events whatever the cell type.
Lab. Biomembranes et Signalisation Cellulaire, CNRS UMR 6558, Université de Poitiers, F-86022 Poitiers, France
ACTIVATION OF ICRAC BY STORE DEPLETION AND RECEPTOR STIMULATION IN FRESHLY ISOLATED RAT HEPATOCYTES
Rychkov G., Litjens T., Roberts M., Barritt G.
Activation of Ca2+-conducting cation channels, or so-called store operated channels, in the plasma membrane in response to depletion of intracellular Ca2+ stores is a universal feature of the Ca2+signalling mechanism in most non-excitable cells. One of the best-known store operated channels, Ca2+release activated Ca2+ (CRAC) channel has been extensively characterised in a number of immortalised cell lines. There is little evidence, however, that ICRAC is activated under physiological conditions in cells in primary culture.
In the current series of experiments we have shown that depletion of the intracellular Ca2+ stores in freshly isolated rat hepatocytes by IP3, thapsigargin or 10 mM EGTA activated an inward current highly selective for Ca2+, which could be blocked by sub-micromolar concentrations of trivalent cations and by 50 micromolar of 2-APB. Changes of the current amplitude with Ba2+ substitution for Ca2+ and the kinetics of the current inactivation at negative potentials were similar to that of ICRAC described in immortalised cell lines. The amplitude of ICRAC in rat hepatocytes varied between -20 and -120 pA at -100 mV, with an average density of about -1 pA/pF. The same current was activated by the application of 20 nM vasopressin or 5-50 micromolar ATP. Activation of ICRAC by 20 nM of vasopressin or 5 microM ATP occurred with a considerable delay (2-4 minutes). While the delay could be shortened using higher concentrations of the agonist, the rate and extent of ICRAC development were largely unaffected.
It is concluded that in rat hepatocytes ICRAC is the major pathway of Ca2+entry in rat hepatocytes, regulated by phospholipase C coupled receptors.
University of Adelaide and Flinders University of South Australia, Australia.
NAADP MAY INTERACT WITH THE RYANODINE RECEPTORS IN THE NUCLEAR ENVELOPE OF PANCREATIC ACINAR CELLS
Gerasimenko J.V., Maruyama Y., Tepikin A.V., Petersen O.H., Gerasimenko O.V.
We have investigated possible functional interactions of Ca2+ release pathways mediated by nicotinic acid adenine dinucleotide phosphate (NAADP), inositol trisphosphate (IP3) and cyclic ADP-ribose (cADPR) in the envelope of isolated pancreatic acinar nuclei. After isolation, the nuclei were loaded with the calcium sensitive dye Mag Fura Red and changes of fluorescence intensity of the dye in the nuclear envelope were monitored using a Leica laser scanning confocal system.
Recently the existence of possible functional interactions between Ca2+ releasing pathways regulated by NAADP, IP3 and cADPR was shown for intact isolated pancreatic acinar cells. We used caffeine to inhibit IP3 receptors (IP3R) in isolated nuclei. We have found that caffeine itself can induce calcium release from the envelope of isolated nuclei loaded with Mag-Fura Red. Subsequent addition of NAADP in the presence of caffeine induced further calcium release.
We have also used ryanodine (100 µM), which is known as an inhibitor of ryanodine receptors (RyR) when used at a high concentration. Ryanodine did not affect IP3 induced responses, but completely prevented caffeine-induced calcium release. Ryanodine also completely inhibited NAADP-induced and cADPR-induced calcium responses.
These data indicate that NAADP is functionally interacting with the RyR without involving the IP3R. We conclude that NAADP-induced Ca2+ release from the nuclear envelope could be explained by direct or indirect functional interaction of NAADP with the RyR
Physiology Dept, Liverpool University, Liverpool, UNITED KINGDOM
MITOCHONDRIAL REGULATION OF THE STORE-OPERATED CALCIUM CURRENT ICRAC
Bakowski D., Parekh A.
Mitochondria are important regulators of store-operated calcium entry under physiological conditions. By taking up some of the calcium that has been released from the stores by InsP3, mitochondria enable the stores to deplete sufficiently for the store-operated calcium current ICRAC to activate. Furthermore, by buffering incoming calcium, mitochondria reduce calcium-dependent slow inactivation of CRAC channels. Recent work suggests mitochondria might have an addition role in regulating ICRAC. The ability of thapsigargin, which depletes stores independently of InsP3 receptors by inhibiting SERCA pumps, to activate ICRAC is compromised by mitochondrial depolarisation. The involvement of mitochondria here is distal to store depletion and kinetic considerations argue against a role for calcium feedback inactivation of ICRAC. To test the latter more directly, we have carried out experiments using barium to carry ICRAC. Barium is not able to trigger calcium-dependent inactivation of ICRAC in RBL cells. We find that barium permeates CRAC channels, with a macroscopic conductance around 70% that of calcium. In weak buffer, no store-operated barium current is seen unless mitochondria are energised. In experiments monitoring divalent cation entry with fura 2, mitochondrial depolarisation suppressed barium influx. Hence calcium-feedback mechanisms do not account for the regulation of ICRAC by mitochondria under these conditions. Surprisingly, barium influx following store depletion in fura 2-loaded cells was smaller than expected from the electrophysiological recordings of ICRAC. We find barium permeates ICRAC in a voltage-dependent manner and depolarises the membrane potential by blocking potassium channels. This combination accounts for the low barium influx seen in fluorescence experiments. Our results suggest that caution is needed in interpreting data that uses barium to monitor calcium influx in non-excitable cells.
Department of Physiology, University of Oxford, Parks Road, Oxford, OX1 3PT, UNITED KINGDOM
VDAC AND APOPTOSIS: REGULATION AND STRUCTURE-FUNCTION RELATIONSHIP
Bordeaux - FRANCE
(abstract not received)