RECOMBINANT NEURAL AGRIN AFFECTS THE EXCITATION-CONTRACTION MACHINERY IN HUMAN MYOTUBES IN VITRO
Lorenzon P., Bandi E., Formaggio E., Jevsek M., Mars T., Fumagalli G., Grubic Z., Ruzzier F.
It is generally accepted that the neural isoform of agrin is a critical molecule for the acetylcholine receptor clustering and/or stabilisation at the endplate. More recently, it has been shown that recombinant neural agrin mimics the synaptogenic effect of motor neurons inducing microprocess formation in uninnervated myotubes. Taking into account other possible unexplored mechanisms of action, we tested if neural agrin could also be involved in the maturation of the excitation-contraction coupling mechanism. Videoimaging experiments were performed on human myotubes which were either: i) cocultured with foetal rat spinal cord explants; ii) aneurally cultured in medium containing purified recombinant chick neural agrin or iii) aneurally cultured in control medium (without agrin). The maturation of the excitation-contraction coupling mechanism was followed by measuring the percentage of cells exhibiting an intracellular calcium transient when depolarised in the absence of extracellular calcium. The percentage of cells characterised by a mature excitation-contraction coupling mechanism was similar in myotubes cocultured (63.59 ± 7.44%; n = 66) or treated with agrin (70.44 ± 5.52%; n = 40). However, this percentage was significantly lower (28.58 ± 10.09%; n = 68) in myotubes cultured in control medium. Our results suggest that, besides other effects, agrin might also be responsible for the motor neuron-controlled maturation of the excitation-contraction coupling machinery. The molecular details by which agrin induces such process remains to be identified.
Dept Physiol Pathol, Trieste, Italy; Dept Med Publ Health, Verona, Italy; Inst Pathophysiol, Ljubljana, Slovenia
IDENTIFICATION OF TRANSMEMBRANE Ca2+ INFLUX IN CHOLINERGIC Ca2+-SIGNALLING IN SALIVARY ACINAR CELLS
Kruglikov I., Fedirko N., Voitenko N.
The functioning of exocrine cells is under strict parasympathetic control, but the information about Ach-induced intracellular Ca2+ release and transmembrane Ca2+ influx remains obscure. Thus, in the present study we investigated the pathways of acetylcholine (Ach) induced Ca2+ signalling in isolated rat salivary acini. Fluorescent calcium measurements were done using fura-2/AM. Application of 5mkM Ach evoked [Ca2+]i transients with the amplitude of 215+/-22 nM (n=59). Second Ach application produced [Ca2+]i transient with the amplitude of 74+/-5% (n=10) from initial Ach response with no subsequent desensitization. Due to Ach ability to be endogenously hydrolyzed by acetylholinesterases (AchE) we did additional control adding the AchE inhibitor neostigmine (1mM). Application of neostigmine together with Ach did not significantly change the amplitude of Ach-induced [Ca2+]i transients (95+/-3%, n=7), thus showing the absence of active AchEs in our preparation. To study the subsets of Ach receptors responsible for generation of [Ca2+]i transients we used potent muscarinic receptor antagonist atropine. Application of Ach in the presence of atropine (10 mM), gave rise to [Ca2+]i transients with the amplitude of 21+/-5% (n=9) from initial Ach response. Ach-induced [Ca2+]i transients after acini preincubation with thapsigargin (500 nM, 20 min) were reduced by 79+/-6% (n=8). Application of 1 mM benzohexonium and 50 mkM tubocurarin (inhibitors of n type AchRs) decreased the amplitude of Ach-response by 26+/-4% (n=7) and 32+/-6% (n=7) respectively. Application of nAchRs agonist cytisine (100 mkM) induced [Ca2+]i transients with the amplitude of 21+/-2% from initial Ach response, these responses were completely blocked by either benzohexonium or tubocurarin. Thus we conclude that transmembrane Ca2+ influx induced by nicotinic receptors activation is present in salivary acinar cells, though activation of mAchRs is the main source for Ca2+ elevation in the cytoplasm.
Bogomoletz Institute of Physiology, Kiev, Ukraine; I. Franko National University of Lviv, Lviv, Ukraine.
ROLE OF CALCINEURIN IN CHRONIC HYPOXIA-INDUCED HYPERTROPHIC RESPONSE OF RIGHT VENTRICLE
Koulmann N., Sanchez H., Letout A., Ventura-Clapier R., Bigard A.X.
Chronic hypoxia leads to pulmonary hypertension, then to right ventricle (RV) hypertrophy. This process is associated with an increased proportion of beta isoform of myosin heavy chains (bMHC). Calcineurin seems to be involved as an hypertrophic transducing factor in cardiac myocytes. This experiment was designed to study the effects of treatment with cyclosporin A (CsA), an inhibitor of calcineurin, on: 1) the RV hypertrophy related to prolonged exposure to hypoxia; 2) the expression of bMHC in RV and left ventricle (LV).Male Wistar rats were exposed to hypobaric hypoxia (500 hPa) for 3 weeks and treated either by CsA (H-CsA) or by placebo (H-P). Their morphological and contractile properties were compared to those of normoxic rats treated either by CsA at the same dose (N-CsA), or by placebo (N-P). The body weight of hypoxic rats was less than that of normoxic rats (-10%, P<0.001). CsA also induced a depressed growth rate (P<0.001 compared with P groups). The hypoxia-induced RV hypertrophy (+139%, P<0.001)) was prevented by CsA treatment, whereas the overexpression of bMHC (+33%, P<0.001) was similar in H-CsA group (non hypertrophic RV) and H-P group (hypertrophic RV). Hypoxia also induced a slight increase in the LV weight normalized to body weight (+16%, P<0.001). CsA treatment did not prevent this response but in contrast induced a subtle hypertrophic process in LV (+16%, +21% for N-CsA and H-CsA, respectively, P<0.001), likely because of its well-known systemic hypertension effect. An increased expression of bMHC was observed in hypertrophic LV (+18%, +28% and +38% in N-CsA, H-P and H-CsA group, respectively).Then calcineurin seems to be involved in the RV hypertrophy in response to hypoxia. An overexpression of bMHC occured in response to the increased workload, independently of the activation of calcineurin. Whether the hypertrophy observed in LV in response to CsA treatment was minimized by calcineurin-induced inhibition has not been examined in this study.
Département des facteurs humains - CRSSA - BP 87 - 38702 LA TRONCHE CEDEX - FRANCE
INTERACTIONS BETWEEN Ca AND MITOCHONDRIA IN NEURONAL AGEING
Xiong J., Toescu EC.
Normal brain ageing is associated with a degree of functional impairment of neuronal activity that might result in a decrease in memory and cognitive functions. The relationship between mitochondrial function and Ca2+ homeostasis was studied in the cerebellum by use of both brain slices and primary neuronal cultures. The main parameters of Ca2+ homeostasis were not different between young and old neurones with the notable exception of a prolonged rate of Ca2+ recovery following neuronal stimulation (either depolarization or glutamatergic). In addition, in aged preparations, significantly more neurones showed an early Ca2+ dysregulation, resulting in neuronal death. The use of simultaneous loading with Ca2+ and mitochondrial membrane potential-sensitive dyes showed that increases in cytosolic [Ca2+]i over a threshold value (400 nM) evoked a mitochondrial depolarization response. In the aged neurones the mitochondria had a significantly longer repolarization response and quantitative analysis showed a direct correlation between the delays in mitochondrial repolarization and [Ca2+]i recovery, indicating the causal relationship between the two parameters. Inhibition of the mitochondrial permeability transition pore had several protective effects: it enhanced the rate of mitochondrial repolarization and Ca2+ recovery and decreased the percentage of neurones showing early Ca2+ dysregulation. Western blot analysis of the expression of several members of the Bcl-2 family showed no difference between young and old cerebella. The present results show that the changes in Ca2+ homeostasis associated with ageing are mainly due to a metabolic dysfunction in which the mitochondrial impairment play an important role.
Dept. Physiology, Birmingham University, UNITED KINGDOM
ACTIVATION OF L-TYPE CALCIUM CHANNELS BY VIP INDUCES PROLACTIN GENE EXPRESSION IN AVIAN PITUITARY
Al-Kahtane A., El-Halawani M.
Our previous work demonstrated that Ca2+ influx through voltage-gated L-type calcium channels mediated the stimulatory effects of Vasoactive Intestinal Peptide)VIP( on prolactin (PRL) gene expression and release in cultured turkey anterior pituitary cells. The objective of this study was to examine the possible involvement of protein kinase C (PKC) in mediating VIP-induced Ca2+ influx and the subsequent stimulation of PRL gene expression and release. The level of PRL gene expression was determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) of PRL-mRNA. The homologous radioimmunoassay (RIA) was used to measure the level of PRL secretion from cultured turkey anterior pituitary cells. The PKC inhibitor bisindolylmaleimide I (BI) significantly (P<0.05) reduced VIP-stimulated PRL mRNA levels. In contrast, incubating the cells with the PKC activator, phorbol-12-mysterate-13-acetate (PMA), resulted in a significant (P<0.01) increase in PRL mRNA levels and PRL release. The stimulatory effects of VIP and PMA were not additive when combined together. Finally, PKC involvement in Ca2+ influx-stimulated PRL expression and release induced by the L-type Ca2+ channel agonist Bay K-8644 was examined. The PKC inhibitors staurosporine (ST) and bisindolylmaleimide I (BI) did not reduce PRL mRNA levels stimulated by Bay K8644. However, PRL secretion stimulated by VIP or Bay K-8644 was significantly (P<0.05) reduced by PKC inhibitors. The results of this study show clearly that: 1) PKC plays a major role in mediating VIP induction of Ca2+ influx through the voltage-gated L-type Ca2+ channels in cultured turkey primary anterior pituitary cells, and 2) PKC-dependent signal transduction pathway contributes to VIP effects on PRL gene expression and PRL release in avian species. Supported by USDA grant # 00-02127.
Teachers College, Riyadh, Saudi Arabia; University of Minnesota, Saint Paul, USA
VASORELAXANT EFFECT OF TOTAL FLAVONES FROM DENDRANTHEMA MORIFOLIUM ON RAT THORACIC AORTA
Jin HF., Shan QX., Jiang HD., Tu J., Xia Q.
Objective: To investigate the vasorelaxant effect of total flavones from the dendranthema morifolium (Ramat.) Tzvel. cv. Hangju (FDM) in rat aortic rings. Methods: The isolated thoracic aortic rings were mounted on the organ bath and the tension of the vessel was recorded. Results: FDM completely relaxed, in a concentration-dependent manner, the contractions induced by either phenylephrine or a high concentration of KCl (60 mmol/L) in endothelium-intact rat aorta. Mechanical removal of endothelium did not significantly modify the vasorelaxant effects of this FDM. In endothelium-denuded aortic rings depolarized by 60 mmol/L KCl, FDM inhibited Ca2+-induced contraction. It also reduced the transient contraction elicited by phenylephrine in Ca2+-free medium, but had no effect on active phorbol ester-induced contraction. Pretreatment of endothelium-denuded aorta with propranolol, a beta-adrenoceptor antagonist, significantly attenuated the relaxant effect of FDM. Conclusion: These results indicate that FDM induces an endothelium-independent relaxation in rat aortic rings. The mechanisms may include the activation of beta-adrenergic receptor, reduction in Ca2+ influx through the voltage-dependent and receptor-operated channels, and inhibition of intracellular Ca2+ release in the vascular smooth muscle cells.
Department of Physiology, Zhejiang University School of Medicine – P.R. CHINA
EFFECT OF STREPTOZOTOCIN-INDUCED DIABETES ON SALIVARY SECRETORY CELL Ca2+-ATPASES
Fedirko N., Vats J., Voitenko N.
Ca2+ pumps regulate [Ca2+]i thus playing an important role in exocytosis of secretory cells. Diabetes is associated with changes in cellular Ca2+ homeostasis and functional disorders in effector’s organs. Particularly, the patients with diabetes mellitus suffered with hypo-salivation but its cellular mechanism is unknown. We assume calcium-dependence of this disorder. Because of that in the present research we studied the influence of diabetes on the Ca2+-ATPases of salivary cells. The study was done on isolated cells and microsomes. Inorganic phosphor (Pi) content was measured using Fiske-Subbarow method. Fluorescent calcium measurements were performed using fura 2/AM. Diabetes was induced by intraperitoneal injection of streptozotocin (80 mg/kg proportion). Animals were taken into experiments 6 weeks after. The glucose concentration was 5-9 and 12-28 mM for normal and diabetic animals respectively. We showed that under diabetic neuropathy the resting [Ca2+]i increases by 66%. This increase could be due to modified functioning of Ca2+ extruding systems. Next we demonstrated that under the diabetes kinetic properties of total Ca2+-ATPase activity are changed: Pmax decreased by 70%, ν0 – by 56% and t – by 67%. Diabetes decreased specific PMCA and SERCA activities by 16±7% and 40±9% respectively. The substrate affinity of PMCA and SERCA of salivary cell under diabetes was also modified: Pmax decreased on 37% and 67%; Km for ATP decreased by 85% and 41% for PMCA and SERCA respectively, Hill’s coefficient for PMCA did not changed while for SERCA it increased by 26%. We suppose that under diabetes lowered activities of PMCA and SERCA are associated with decreased amount of active molecules and/or enzyme rotation. Decreased Km and Hill’s coefficient under diabetes testify about the enhanced affinity of Ca2+,ATP-ases to the lowered cellular ATP concentration that could be a physiological protection from their suppressed activity. Supported by CRDF grant to NV.
Ivan Franko National University of Lviv, Lviv, Ukraine; Bogomoletz Institute of Physiology, Kiev, Ukraine
S2 New aspects of ionic transport (I) The Pflugers Archiv Symposium
EPITHELIAL SODIUM CHANNELS: LESSONS FORM HUMAN DISEASES AND MOUSE MODELS
According to the hypothesis put forward by Guyton, over 20 years ago , control of blood pressure at steady state and on a long-term basis is critically dependent on renal mechanisms. A number of genes expressed in various parts of the nephron have been shown to be directly involved in the control of blood pressure. The identification of mutations in monogenic diseases such as the Bartter’s or the Gitelman’s syndromes clearly indicate that defects in ion transporters expressed in the thick ascending limb (TAL) or in the distal convoluted tubule (DCT) may lead to a severe salt-loosing syndrome with a hypotensive phenotype. In the Aldosterone-Sensitive Distal Nephron (ASDN) i.e late distal convoluted tubule (late DCT), the connecting tubule (CNT), the cortical collecting duct (CCD) and, to some extent, the outer medullary collecting duct (OMCD) and inner medullary collecting duct (IMCD), the final control of sodium reabsorption is achieved through an amiloride-sensitive electrogenic sodium reabsorption which is under tight hormonal control, aldosterone playing the key role. The main limiting factor in sodium reabsorption in this part of the nephron is the apically located amiloride-sensitive epithelial sodium channel (ENaC). Two monogenic diseases have been linked to ENaC subunit genes; first, pseudohypoaldosteronism Type 1, a severe autosomal recessive form of a salt-loosing syndrome is due to loss (or partial loss) of function mutations in the a, b or g subunit genes of ENaC. Gain of function mutations in the b or g subunit of ENaC lead to a hypertensive phenotype (Liddle syndrome), a paradigm for salt-sensitive hypertension.
In this presentation, I will discuss conditional gene targeting experiments that offer new opportunities to study in vivo ENaC function in the collecting duct.
Institut de Pharmacologie et de Toxicologie,Université de Lausanne, Lausanne, Switzerland
THE FAMILY OF EPITHELIAL CALCIUM CHANNELS
The recent expression cloning of the epithelial calcium channels, TRPV5 and TRPV6 has provided a molecular basis to explore the characteristics of the rate-limiting entry step in transcellular calcium (re)absorption. These channels are primarily expressed in the distal part of the nephron, proximal small intestine and placenta, organs that play a key role in calcium homeostasis of the body. These channels of about 730 amino acids contain 6 putative membrane-spanning domains with an additional hydrophobic stretch predicted to be the pore region. TRPV5/6 resemble the recently cloned capsaicin receptor and the transient receptor potential-related ion channels with respect to its predicted topology. In kidney, TRPV5/6 are abundantly present in the apical membrane of calcium transporting cells and colocalize with 1,25-dihydroxyvitamin D3-dependent calbindin-D28K, sodium-calcium exchanger and plasma calcium ATPase. Several studies in animal models demonstrated that TRPV5/6 expression in kidney and intestine is positively controlled by the important calciotropic hormone, 1,25-dihydroxyvitamin D3. TRPV5/6 expression in eukaryotic cells confers calcium influx with properties identical to those observed in native distal renal cells including a high calcium selectivity and negative feedback regulation to prevent calcium overload during transepithelial transport. TRPV5/6 are co-expressed in several tissues forming homo- and heterotetrameric channel complexes with distinct channel properties. Consequently, regulation of the relative expression levels of TRPV5/6 may be a mechanism to fine-tune the calcium transport kinetics in TRPV5/6-expressing tissues. The S100A10 and annexin complex is an important regulator determining the plasma membrane localization of these channels. In conclusion, TRPV5 and TRPV6 constitute a new family of calcium channels with the expected properties for being the gatekeepers of 1,25-dihydroxyvitamin D3-dependent active calcium (re)absorption.
Cell Physiology, University Medical Centre Nijmegen, the Netherlands
INVOLVEMENT OF AN ANION EXCHANGER IN REGULATORY VOLUME DECREASE (RVD).
Borgese F., Gabillat N., Guizouarn H.
Trout erythrocytes possess multiple swelling-sensitive transport pathways: a KCl cotransport and an osmolyte channel permeable to diverse solutes (taurine, Na+ and K+Cl independent). This channel of broad specificity is activated by a decrease in intracellular ionic strength independently of the magnitude of cell swelling. The anion exchanger AE1 (also termed band 3) is a major constituent of erythrocyte plasma membrane. The particular sensitivity of the swelling-sensitive osmolyte channel to a wide range of drugs known as potent inhibitors of band 3 protein has prompted the suggestion that AE1 might be involved in volume regulation.
Indeed, when expressed in Xenopus oocytes, the trout red blood cell anion exchanger (tAE1) elicits, as expected, an anion exchange activity. But simultaneously tAE1 expression results in the appearance of both an anion conductance and a transport of taurine and cations. tAE1 forms an organic osmolyte channel of broad specificity, having a significant cation permeability. These permeabilities are expected if tAE1 serves as a route for volume regulatory efflux of osmolytes. In contrast, the homologous AE1 from mammalian erythrocytes are devoid of such volume-regulatory functions.
To define the structural domains involved in induction of the channel activity, chimeras have been done between trout and mouse AE1. Results have shown that only the spanning domain of tAE1 is linked to the channel activity and more precisely helices 6, 7, 12 and 13 are required for this function.
It remains to be shown whether other mammalian AE (e.g. AE2 and AE3) expressed in cell types regulating their volume, also possess a channel activity that may be involved in volume regulation.
LPMC. UMR 6078, CNRS – UNSA. 284 chemin du lazaret. 06230 Villefranche sur mer, France.
STRUCTURAL DOMAINS INVOLVED IN SUBSTRATE SELECTIVITY IN TWO NEUTRAL AMINO ACIDS TRANSPORTERS
Soragna A., Valli E., Castagna M., Mari S., Giovannardi S., Bossi E., Peres A.
Two high homologous Na+/Cl-dependent neutral amino acid transporters: KAAT1 and CAATCH2 cloned from the midgut epithelium of the larva Manduca sexta are useful tools to study protein domains involved in substrate selectivity. The ability of the two proteins to transport different amino acids depends on the cotransported ion, on pH and on the membrane voltage. Each organic substrate gives rise to transport-associated currents with its own characteristics, which are notably distinct between the two proteins. Differences in amplitude, kinetics and voltage-dependence of the transport-associated currents have been observed especially in the presence of the amino acids leucine, methionine, threonine and proline. These diversities were used to investigate the structural determinants involved in the substrate selectivity. To identify these protein regions, four chimera proteins between the two transporters were built.The high homology let us to exchange different fragments of the protein without introducing mutations. The chimera proteins obtained, heterologously expressed in Xenopus laevis oocytes were analysed by two-electrode voltage clamp and uptake measurements.The proteins where the first three domains were exchanged, C3K9 and K3C9 show electrophysiological characteristics and uptake of [H4]leucine and [H4]proline of KAAT1 and CAATCH2 respectively. These first results show that the transmembrane domains (TMs) 1-3 in KAAT and CAATCH are not involved in organic substrate selectivity. Consequently the substitution of the last four domains in C3K9 and K3C9 giving the proteins C3K5C4 and K3C5K4 shows again that these proteins have the same behaviour of KAAT1 and CAATCH2 in electrophysiological and transporter determination. We can conclude that in KAAT1 and CAATCH2 only the central TMs(from 4 to 8)of the protein is responsible of the substrate selectivity.
DBSF-University of Insubria, Varese Italy- Inst. of Biochemistry and General Physiology, University of Milan- Italy
DIACYLGLYCEROL DIRECTLY ACTIVATES A NON-SELECTIVE CATION CHANNEL IN DEDIFFERENTIATED CARDIOMYOCYTES
Guinamard R., Lenfant J., Bois P.
In the adult rat cardiomyocytes culture, proposed as an in vitro myocardial hypertrophy model, we have recently characterized a rise of the density of a calcium-activated non-selective cation channel (NSCCa) during dedifferentiation. The channel was selective for Na+ and K+ and impermeable for Ca2+ ions. It had a conductance of 20 pS in the inside-out configuration and was activated by rise in internal Ca2+. A pre-stimulation by ATPgS or by a phorbol ester increased the channel detection, suggesting that PKC is involved in the regulation of NSCCa channels.
Here we reported the regulation of the channel by DAG analogues and PKC. In cell-attached configuration, it had a conductance of 20.2 pS and a reversal potential of +24 mV (n=7) (pipette and bath, 140 mM NaCl). Application of the permeable DAG analogue OAG (0.1 mM) or the PKC activator PMA (500 nM) increased the open probability (Po) from 0.06 to 0.55 (n=4) and from 0.05 to 0.46 (n=4) respectively. In the presence of the PKC inhibitor Calphostin C (0.001 mM), OAG still had an activating effect while PMA had no effect. In inside-out configuration, DAG analogues OAG (0.1 mM) or SAG (0.01 mM) applied to the inside of the membrane increased Po from 0.10 to 0.59 (n=6) and from 0.07 to 0.65 (n=9), respectively. We infer that the NSCCa channel is under the control of DAG via the PKC pathway but also via a direct interaction.
In models of hypertrophy it was shown that DAG contents and PKC activity increase during hypertrophy, that would increase channel activity. Thus the NSCCa channel is a candidate for the genesis of arrhythmias in ventricular cells. In addition, this new regulation of the channel by DAG and PKC could help to understand the physiological role of the NSCCa channels family.
CNRS UMR 6558, Université de Poitiers – France
20-HETE INOTROPIC EFFECTS INVOLVE THE ACTIVATION OF NON-SELECTIVE CATIONIC CURRENT IN ASM
Rousseau E., Cloutier M., Campbell S., Basora N., Proteau S., Payet M.D.
Eicosanoids are important lipid mediators. 20-hydroxyeicosatetraenoic acid (20-HETE) controls several mechanisms such as vasoactivity, mitogenicity and ion transport in various tissues. Our goal was to quantify the effects of 20-HETE on the tone and electrophysiological properties of airway smooth muscle (ASM). Isometric tension measurements, performed on guinea pig ASM, showed that 20-HETE induced a dose-dependent inotropic effect, with an EC50 value of 1.5 µM and a Hill coefficient of 0.77. The sustained contraction, requiring Ca2+ entry, was partially blocked by 100 µM Gd3+ and 1 µM nifedipine, revealing the involvement of non-capacitative Ca2+ entry and L-type Ca2+ channels, respectively. Microelectrode measurements showed that 3 µM 20-HETE depolarized the membrane potential in guinea pig ASM by 13 ± 2 mV (n = 9). Depolarizing effects were observed in absence of epithelium as well as in the presence of OAG (a PKC and TRP channel activator ). Patch clamp recordings demonstrated that 1 µM 20-HETE activated a non-selective cationic inward current which might be support by the activation of TRP channels. The presence of the TRPC mRNA was confirmed by RT-PCR in guinea pig ASM cell. Together our results suggest that an eicosanoid, such as 20 HETE, might activate a non-selective cationic current generated by a member of the TRP channel-receptor family. Supported by the CIHR
Physiology and Biophysics, Fac. of Medicine,Sherbrooke, QC , Canada J1H 5N4
TRPV CHANNELS: STRUCTURE – FUNCTION RELATIONSHIP AND PROMISCUOUS GATING BEHAVIOUR
Calcium signals control a plethora of short- and long-term cell functions. In most non-excitable cells, sustained entry of extracellular calcium upon various stimuli essentially contributes to those Ca2+ signals. Molecular candidates for this entry are cation channels of the “transient receptor potential” (TRP) superfamily. Activation of TRP channels, consisting of three subfamilies (TRPC, TRPV, TRPM), is still very little understood. Examples of activation of TRP channels from all three subfamilies will be discussed. Main focus is on the members of the TRPV subfamily, among which the TRPV4 channel shows a surprising gating promiscuity. It can be activated by cell swelling, heat, or phorbol esters. Endogenous activators of the channel have not yet been described. It will be shown that arachidonic acid (AA) is a robust activator of TRPV4 which may also explain activation of TRPV4 by the endocannaboids anandamide and 2- arachidonyl glycerol which likely requires metabolisation to AA. Lipid messengers downstream of arachidonic acid might act as endogenous TRPV4 activators. For TRPV5 and 6, the only highly Ca2+ - selective channels within the TRP super-family, a voltage dependent gating mechanism will be discussed, which includes an open pore block by Mg2+ and a highly Ca2+ - sensitive mechanism of inactivation. Regulation of channel availability by interaction with a protein bound to the C-terminus of both channels will be demonstrated. Functional consequences of these different mechanisms of gating will be discussed.
KU Leuven, Campus Gasthuisberg, Department of Physiology, B-3000 LEUVEN, Belgium
PHYSIOLOGY AND PATHOPHYSIOLOGY OF CHLORIDE TRANSPORT
Mouse models and human genetic diseases have recently shed considerable light on the function of CLC chloride channels and KCC K-Cl-cotransporters. This talk will focus on three pathologies, all affecting the kidney and two of them the inner ear.
ClC-5 is an endosomal chloride channel that is essential for the acidification of proximal tubular endosomes by providing an electrical shunt for the proton pump. Its disruption leads to a defect in endocytosis. This leads to secondary changes in calciotropic hormones (PTH and VitD) which eventually lead to kidney stones in Dent's disease.
ClC-K channels, by contrast, are plasma membrane channels involved in transepithelial transport. They need barttin, a small beta-subunit, for their transport to the surface. Mutations in ClC-Kb lead to Bartter syndrome type III and in barttin to Bartter syndrome IV that also includes deafness. The importance of these channels in ion transport in the kidney and the stria vascularis of the cochlea will be discussed.
KCC4 is an electroneutral K-Cl cotransporter that is e.g. expressed in renal proximal tubules and intercalated cells, and, in Deiter's cells that support outer hair cells in the inner ear. Its disruption in mice leads to deafness that is associated with renal tubular acidosis. This pathology will be compared to that of the Bartter syndrome type IV.
ZMNH, Universität Hamburg, Germany
MODULATION OF ION CHANNELS BY ESTROGENS
Estrogen and antiestrogens are capable of rapid modulation of Maxi Cl- and Maxi K+ channels in vascular smooth muscle cells (Valverde et al, 1999; Díaz et al. 1999). The mechanism of action leading to the modulation of these channels seems to be different. Modulation of Maxi K+ involves a direct interaction between the hormone and the channel complex, as well the participation of second messengers. Rapid modulation of vascular smooth muscle ion channels by estrogens leads to endothelium-dependent and independent vasodilatation. A key player in the control of vascular smooth muscle tone is the Maxi-K channel. This channel consists of two subunits: a pore forming a subunit and a regulatory b subunit which confers the channel with a higher Ca2+ sensitivity. We have recently described the modulation by 17b-estradiol of both native and heterologously expressed Maxi-K channels and found that oestradiol activates the channels through its interaction with the b subunit (Valverde et al. 1999).
Maxi Cl- channels have been recorded in many different cell types. We have described their modulation by estrogens and antiestrogens in vascular smooth muscle and neuroblastoma cells (Diaz et al. 1999, 2001), a process that requires the generation of intracellular signals, although its relevance to cell physiology remains unknown.
Our results suggest that estrogen and antiestrogens exert different rapid actions on the same cell type, an observation that fits the current view of multiple sites of action for estrogens (Nadal et al. 2001).
Díaz, M. et al (1999). Journal of Physiology 517.P, 7S-8S.
Diaz et al. (2001) Journal of Physiology 536.1, 79-88.
Nadal, A. et al (2001). News in Physiological Science, 16, 251-255.
Valverde, M.A. et al. (1999). Science, 285, 1929-1931.
This work was funded by Human Frontiers Science Program y Distinció de la Generalitat de Catalunya.
Universitat Pompeu Fabra, Barcelona, Spain
EXHALED NITRIC OXIDE AS A MARKER OF ION TRANSPORT IMPAIRMENT IN CYSTIC FIBROSIS PATIENTS
Texereau J., Fajac I., Hubert D., Dusser D., Bienvenu T., Dall Ava-Santucci J., Dinh-Xuan A.
Because production of nitric oxide – a key molecule which regulates many important physiological functions of the airways – is reduced in cystic fibrosis, a condition characterized by defective ion transport, we aimed to test the hypothesis whether reduced nitric oxide production might affect airway ion transport in vivo in cystic fibrosis patients.
Pulmonary function, nasal potential difference and exhaled nitric oxide were measured in sixty adults with cystic fibrosis. A slope of lung function decline was determined retrospectively for each patient using simple linear regression with all available spirometric values obtained over a time period of five years that preceded patient’s entry in the study.
The annual rates of decline in forced expiratory volume in one-second were directly correlated to abnormal nasal potential difference values (P<0.05). The latters were inversely related to exhaled nitric oxide concentrations (P<0.01). Cystic fibrosis patients with normal nasal potential difference had higher exhaled nitric oxide concentrations (20.1 ± 2.6 parts per billion) than healthy controls (12.1 ± 0.8 parts per billion, P<0.01) whose exhaled nitric oxide concentrations were significantly higher than those of cystic fibrosis patients with abnormal nasal potential difference (8.6 ± 0.5 parts per billion, P<0.01).
These data suggest that exhaled nitric oxide is related to nasal transepithelial potential difference and that nitric oxide could play a compensatory role on defective cystic fibrosis transmembrane conductance regulator protein activity.
Hopital COCHIN, Paris, FRANCE
ANION SELECTIVITY AND GATING OF TORPEDO CLC-0 CHLORIDE CHANNEL
Bennetts B., Roberts M., Bretag A., Rychkov G.
Members of the ClC family are ubiquitous, Cl specific channels that are expressed in both outer membranes and the membranes of intracellular organelles. The muscle-type ClC channels ClC-0 and ClC-1 are activated by membrane depolarisation, allowing Cl to enter the cell and repolarise the membrane. Activity of these channels is sensitive to Cl concentration in the external solution, and so they have been referred to as Cl activated Cl channels.
In the current experiments a series of anions was used to probe the permeation pathway and gating of ClC-0. Equilibrium selectivity for various anions, determined from reversal potential measurements corresponded to a moderately strong field site in the pore, and was similar to ClC-1. The selectivity of the site that regulates activity of the channel appeared to be different in the open and closed states, such that in the closed state the regulatory site specifically bound Cl in preference to larger anions such as ClO3 and ClO4, but in the open state these ions could block Cl conductance by binding to the regulatory site. Selectivity sequence and the relative ability of different anions to affect fast gating determined in the present study implied a smaller size of the pore and smaller dimensions of the regulatory Cl binding site in ClC-0 compared to ClC-1. Linear free energy relationships analysis suggested that the conformational changes at the regulatory site during open-closed transitions coincided with voltage sensation by the channel.
University of Adelaide, and University of South Australia, Australia.
SODIUM AND CALCIUM CURRENTS IN CHICK EMBRYO DEVELOPING TYPE I AND TYPE II HAIR CELLS
Bosica M., Zucca G., Valli P., Masetto S.
By using the whole-cell patch-clamp technique in combination with the chick embryo crista slice preparation, we have recorded inward ionic currents from type I and type II hair cells in situ, at different stages of development. To block outward K+ currents, KCl in the pipette solution was substituted by NMDG and CsCl. Voltage-clamp experiments showed that a large fraction of type I and type II hair cells express a sodium current (INa), from embryonic day 14 (E14) up to hatching (E21). INa activated around –60 mV, peaked around –20 mV, displayed fast activation and inactivation, and was completely, and reversibly, blocked by tetrodotoxin (TTX; Kd = 3 nM). A peculiar property of INa concerned its steady-state inactivation, in that it was complete at –60 mV (V1/2 = -96 mV).
A very small sustained inward current was also present at voltages less negative than –60 mV, from the first developmental stage investigated (E10). This current was not blocked by TTX, whereas it was completely abolished by Cd++ 100 μm, which also blocked INa. The sustained inward current was increased by perfusing an extracellular solution containing Ba++ instead of Ca++. INa conversely was reduced by Ba++ perfusion. IBa was present in all hair cells investigated. It activated around –60 mV and peaked around –20 mV. It showed rapid activation and little inactivation. Its time- and voltage-dependent properties appeared similar in type I and type II hair cells. This current has been identified as a Ba++ current flowing through voltage-dependent Ca channels.
Ca channels are involved in afferent synaptic release from the basal pole of the hair cells. Present results suggest a similar operational range for Ca channels in type I and type II hair cells. Na channels on the other side could reinforce, at least in a subpopulation of hair cells, membrane depolarization and thus boost synaptic output.
Dipartimento di Scienze Fisiol. - Farmacol. - Università di Pavia – Italy
A NOVEL VOLTAGE-DEPENDENT CHLORIDE CURRENT ACTIVATED BY EXTRACELLULAR ACIDIC pH IN RAT SERTOLI CELLS
Auzanneau C., Thoreau V., Norez C., Becq F.
Sertoli cells from mammalian testis are involved in development and maintenance of spermatogenesis, support and nourishment of germ cells and synthesis and release of several proteins and a potassium-rich fluid into the lumen of seminiferous tubules. Sertoli cells express a variety of ionic channels among them voltage-dependent Ca2+ and calcium-dependent Cl- channels. Using whole-cell patch clamp experiments and iodide efflux, a novel chloride current was identified. It is activated only in the presence of an extracellular acidic-pH with an estimated half maximal activation at pH 5.5. The current is strongly outwardly rectifying, activated with a fast time-dependent onset of activation but a slow time-dependent kinetic at depolarization pulses. The pH-activated chloride current was not detected at physiological or basic pH, and is not sensitive to intracellular nor extracellular Ca2+ variation. The pharmacology of this channel has been established by iodide efflux. Its anionic selectivity was Cl->Br->I->gluconate. We have performed an RT-PCR analysis to search for voltage-dependent chloride rCLC channels in cultured rat Sertoli cells. Among the nine members of the family only rClC-2, rClC-3, rClC-6 and rClC-7 have been identified. The inwardly rectifying rClC-2 chloride current was activated by hyperpolarization but not by pH variation. A different depolarization-activated outwardly rectifying chloride current was activated only by hypotonic challenge and may corresponds either to rClC-3 or rClC-6. Immunolocalization experiments demonstrate that rClC-7 resides in intracellular compartment of Sertoli cells. This study provides the first functional identification of a native acid-activated chloride current. Based on our molecular analysis of rClC proteins, this new chloride current does not corresponds to rClC-2, rClC-3, rClC-6 nor rClC-7 channels.
Supported by le Conseil régional du Poitou-Charentes.
LBSC, UMR 6558, Université de Poitiers, 86022 Poitiers, France
NASAL POTENTIAL DIFFERENCE MEASUREMENT IN CYSTIC FIBROSIS AND ELECTRONIC DATA ACQUISITION
Ergonul Z., Yilmaz G., Balkanci ZD., Kiper N., Yalcin E., Dogru D., Ozcelik U., Gocmen A.
Objectives: Cystic fibrosis (CF) patients demonstrate more a negative potential difference on respiratory epithelia than normal controls. The CF gene product, cystic fibrosis transmembrane conductance regulator (CFTR), is a chloride channel that also acts as a regulator of heterologous ionic channels. Abnormalities of ion transport in respiratory epithelia of patients are associated with enhanced sodium absorption and defective cAMP mediated chloride secretion, which contributes to the dehydration of airway secretions. Transepithelial nasal potential difference (NPD) measurement has been used as a diagnostic test for CF. NPD measurement techniques commonly vary between centers. It has been shown that there are large differences in reproducibility of measurements between different study sites. In order to standardize measurement protocols, voltmeter input impedance standardizations and electronic data acquisition have been suggested.
Methods: In our study NPD was measured using an adaptation of the method described by Alton which involves an epicutaneous reference electrode and intranasal placement of an exploring electrode with a Foley catheter. The DA100B differential amplifier module of MP100 computer based data acquisition and analysis system replaced a high impedance voltmeter. Our study included 40 CF patients (18 females, mean age 9.3 yrs, range 2-20 yrs) and 37 controls (17 females, mean age, 17.08 yrs and range 2-34 yrs).
Results: The CF group NPD was significantly higher (mean ± SEM, -39.21 ± 1.74 mV) than that of controls (-18.24 ± 1.48 mV, P< 0.00001). No significant difference was noted between left and right sides in all groups. Neither the age nor sex of the subject influenced the measurements.
Conclusion: Our results are consistent with the published data. We suggest that MP-100 system provides suitable monitoring and recording during measurements, which could eliminate the bias due to different voltmeters and hand analysis.
Department of Physiology and Department of Pediatrics, Hacettepe University, Faculty of Medicine, Ankara, Turkey
SINGLE-CHANNEL Cl- CURRENTS IN INSIDE-OUT PATCHES FROM BROWN FAT CELL MEMBRANES
Sabanov V., Cannon B., Nedergaard J.
The function of brown adipose tissue is sympathetically regulated heat generation. Norepinephrine induces in brown adipocytes a very fast and dramatic activation of metabolism, which is accompanied by complex electrical perturbations in the plasma membrane. The very first component of this electrical response has been shown to be due to Cl- efflux. The present investigation was designed to explore the Cl- permeability of the membrane at the single-channel level. Earlier we described Cl- channel currents in the inside-out configuration that can be promoted by strong depolarisation of the excised patches, although could not be observed in the cell-attached mode (Sabanov & Nedergaard, 1995, BBRC, pp.639-647). They have relatively unstable amplitude and reveal multiple closed and open states and burst/gap behaviour. In an effort to characterize the channels functionally we examined their calcium dependence. According to their behaviour in Ca2-free solutions containing Ca-chelator (EGTA or BAPTA), the currents (channels?) can be divided into two groups. The average current amplitudes and, correspondingly, mean single-channel conductivities in these groups are different. The activity of the “large” (~59 pS) channels was completely blocked by Ca-free solutions, whereas in the “small” channels (~38 pS) a more specific rearrangement occurred. The possible explanation is that in either case both EGTA/BAPTA and Ca2+ can inhibit the channel activity. Therefore, in the Ca-free solutions these two factors – presence of chelator and absence of Ca, counteract. In the large channels the blocking effect of the chelator dominated over the stimulating effect of calcium absence; in the second group these two effects were much more equal and mask each other. On the bases of all-point amplitude histogram analysis a double–barrelled structure for both the “large” and “small” channels can be suggested.
The Wenner-Gren Institute, Stockholm University, The Arrhenius Laboratories F3, SE-106 91 Stockholm, Sweden
PROPERTIES OF A CHLORIDE CHANNEL AT THE BASOLATERAL MEMBRANE OF THE MOUSE CONNECTING TUBULE
Nissant A., Teulon J.
We investigated the properties of basolateral Cl- channels on microdissected connecting tubules (CNT) isolated from collagenase-treated mouse kidneys, using the cell-attached and excised configurations of the patch-clamp technique. The bath solution contained (in mM): 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, 10 HEPES (pH 7.4). The pipette solution was similar except for NaCl (145 mM) and KCl (no KCl included).
In the cell-attached configuration, we recorded one channel, which had a linear i/v relationship with a unit conductance of 10.5 +/- 1.2 pS (n = 6, means +/- SEM) and a reversal potential (Er) close to zero (0.8 +/- 5.3 mV). Upon excision, it was possible to assess the anionic selectivity of the channel despite frequent channel rundown by changing the solution on the intracellular side. With a bath NaCl concentration of 14 mM, conductance and Er were 9.8 +/- 0.3 pS and -41.3 +/- 2.6 mV (n = 6), respectively. The PNa/ PCl ratio was 0.08 +/- 0.02 (n = 6). We also investigated the relative permeabilities for halides and nitrate. We obtained relative permeabilities of 0.44 +/- 0.07 (n = 5) for Br-, 0.56 +/- 0.09 (n = 5) for NO3-, 0.77 +/- 0.08 (n = 3) for I- and 0.17 +/- 0.04 (n = 6) for F-. Thus the channel had the relative permeability sequence Cl- ~ I- > Br- ~ NO3- > F-.
The properties of the channel described here are similar to those of a Cl- channel that we have previously described in the basolateral membrane of the mouse DCT.
UMR 7134 CNRS-Université Paris 6, Paris, France.
NEUROHYPOPHYSIAL HORMONE REGULATION OF Cl- SECRETION IN CULTURED GILL CELLS:PRESENCE OF V1 RECEPTORS.
Avella M. (1)., Guibbolini M.E. (2)
Neurohypophysial hormone receptors were studied in primary cultures of sea bass gill respiratory-like cells grown on permeable supports.
Under control conditions, the cultured monolayered epithelium had a short-circuit current (Isc) of 3.5±1.1µAxcm-2. This current had previously been identified as an active Cl- secretion. Addition of increasing concentrations of the fish neurohypophysial hormones, arginine vasotocin (AVT) or isotocin (IT), elicited a concentration-dependent stimulation of the Isc. Maximal increases of 61±12% and 118±28% above the basal Isc value were obtained for 10-7M AVT and IT, respectively. Half-maximal effects were obtained for 3.1x10-9M AVT and for 1.4x10-9M IT. Mucosal application of 1mM DPC (a specific blocker of Cl- channels) revealed a correlation with a hormone-dependent Cl- transport.
Specific V1 or V2 analogues of vasopressin (mammalian hormone) were used to characterize pharmacologically the type of neurohypophysial hormone receptors. While the V1 agonist stimulated the basal Cl- secretion with a similar profile to that of AVT or IT, the V2 agonist had no effect. The V1 antagonist used at a concentration of 5x10-7M totally reversed the 10-8M AVT-stimulated Cl- secretion, whereas the V2 antagonist used at the same concentration had no significant effect. In contrast, similar experiments carried out in the presence of 10-8M IT showed that both antagonists significantly reduced the IT-stimulated Cl- secretion, with an efficiency of the V1 antagonist significantly greater than that of the V2.
This study provides evidence for a neurohypophysial hormone control of Cl- secretion in fish cultured gill respiratory cells. It suggests on physiological basis that the hormonal effect is shared by the two peptides present in fish neurohypophysis, acting by means of two distinct, although pharmacologically similar, V1-type receptors. These specific receptors are expected to play an important role in controlling ion homeostasis in seawater fish
(1)Lab. Physiologie Cellulaire Moléculaire, UMR CNRS 6548, (2)Lab. R.O.S.E., UMR INRA 1112, UNSA, 06108 Nice, France.