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P23-09

CHARACTERIZATION OF DIFFERENT A561 HERG MUTATIONS ASSOCIATED WITH VARIOUS FORMS OF LONG QT SYNDROME

Bellocq C., Louerat-Oriou B., Schott J.J., Le Marec H., Escande D., Baro I.

HERG channels underlie the rapidly activating delayed rectifier potassium current (IKr) in cardiac myocytes. HERG mutations have been linked to congenital long QT syndrome (LQT2) and to acquired long QT syndrome (ALQT).

We have recently identified in a French family a missense mutation (A561P) in the S5 region of HERG. This mutation was not associated with any phenotype until the carrier, a healthy young boy, had arrhythmia while being treated with clobutinol, an antitussive drug (ALQT mutation). Interestingly, two other mutations (A561V and A561T) at this position had already been described. Both were associated with LQT2. In order to address how mutations at the same site in HERG can be related with different phenotype severity, we examined the mechanisms for HERG channels dysfunction in the A561 mutations using electrophysiological methods and immunolocalization experiments in transfected COS-7 cells. We showed that: (i) clobutinol blocks WT HERG current in a dose-dependant manner with a half-maximum block concentration (IC50) of micromolar range. (ii) All mutations lead to channel loss-of-function due to defect in protein traffic towards plasma membrane. (iii) The mutated proteins have a dominant-negative effect on WT HERG, reducing the current by 70%. (iv) Co-expression of WT and A561P (ALQT) HERG induces a 10 mV shift, towards negative potentials, of the activation/V curve whereas co-expression of WT and A561T or A561V (LQT2) proteins does not modify the current voltage-sensitivity. This earlier activation is responsible for an earlier contribution of IKr current during cardiac action potential.

We conclude that the long QT syndrome is a disease of increasing complexity based on many mechanisms involving channel function alteration, abnormality in protein processing and trafficking, and channel pharmacological properties. Interactions between those mechanisms are likely to be determinant for understanding the pathophysiology of LQT2.

INSERM U533, Nantes, France

P23-10

ELECTROPHYSIOLOGICAL EFFECT OF TERIKALANT IN DOG CARDIAC MUSCLE

Biliczki P., Virág L., Iost N., Biliczki A., Gyula Papp J., Varró A.

The class III antiarrhythmic agent RP 58866 and its active enantiomer, terikalant was generally considered as a selective blocker of the inward rectifier K+current, Ik1. These drugs have demonstrated efficacy in experimental arrhythmias, suggesting that block of Ik1 may be a useful antiarrhythmic mechanism. However the selectivity of terikalant on Ik1 is uncertain, especially at high concentration.

Therefore the aim of the present study was to investigate the in vitro electrophysiological effects of terikalant in canine isolated ventricular muscle and Purkinje fibers by applying the standard microelectrode technique.

At a stimulation cycle length of 1000 ms, terikalant (1-5-10-20 µM) significantly prolonged the action potential duration (APD) in papillary muscle. In Purkinje fibers terikalant (2.5 µM) produced a marked APD prolongation. At concentration <5µM terikalant lengthened APD in a reverse frequency-dependent manner both in papillary muscle and Purkinje fibers. At concentrations higher than 5 µM the APD prolongation was not clearly rate dependent. In right ventricular papillary muscle terikalant concentrations equal or higher than 5 µM depressed the maximal upstroke velocity of the action potential (Vmax) in a frequency dependent manner (at CL=300-5000 ms) The onset kinetics of the terikalant induced Vmax block was rapid (2.5 beat-1) like Class I/B, and the offset (recovery) kinetics of terikalant induced Vmax block was intermediate (1377.4 ms) between Class I/A and Class I/B.

Terikalant, like other class III antiarrhythmic drugs, at concentration lower than 5 µM has reverse rate dependent effect on the repolarization but at higher concentration also blocks the inward sodium current (INa) in a use-dependent manner.

Dept. of Pharmacology and Pharmacotherapy, University of Szeged, Faculty of Medicine, Szeged, Hungary

P23-11

THE ANNEXIN II SUBUNIT P11 IS ASSOCIATED WITH THE K2P POTASSIUM CHANNEL TASK1

Girard C., Tinel N., Terrenoire C., Romey G., Lazdunski M., Borsotto M.

Background potassium conductances control the resting potential and input resistance of cells, two key components of neuronal excitability. The channels responsible for these background currents form a new family of potassium channels. Open at rest, these channels (K2P channels) lack both voltage and time depedencies. K2P channels are insensitive to the classical K+ blockers, and are regulated by physical and chemical stimuli including pressure, temperature, lipids, pH, and neurotransmitters. Among these channels, TASK1 possesses a C-terminus PDZ consensus binding site (SSV). In heterologous expression systems, wild type (wt) TASK1 is active whereas the deleted SSV mutant (TASK1*SSV) is inactive. In order to identify proteins associated with the TASK1 PDZ motif, we used the yeast two hybrid system to screen a heart cDNA library with the C-terminus of TASK1 as bait. We found that p11, the light chain of annexin II, interacts with TASK1. This interaction, fully reproduced in vitro using GST Pull-down as well as immunoprecipitation techniques, is specific and requires the integrity of TASK1 PDZ motif. By immunocytochemistry we showed that TASK1 wt is located at the plasma membrane in transiently transfected COS cells, whereas TASK1*SSV is not. Successive deletion of TASK1 C-terminus allowed us to identify a retention motif, immediately upstream the PDZ motif, corresponding to the KRR sequence. We propose that during synthesis, partially folded or misfolded TASK1 subunits are retained in the ER through the binding of ER resident proteins to their retention motif. Correctly folded TASK1 channels expose their PDZ motif which interacts with p11, masking the retention motif and allowing the forward transport of the channel to the plasma membrane. Since TASK1 contributes to the resting membrane potential of various neuronal cells, genetic defects disrupting the SSV motif of TASK1 could induce drastic changes in neuronal excitability, leading to pathological states.

Institut de Parmacologie Moleculaire et Cellulaire, Sophia Antipolis, France

P23-12

SIGMA LIGANDS INHIBIT TUMOR CELL PROLIFERATION THROUGH K+ CHANNEL MODULATION AND P27 ACCUMULATION

Renaudo A., Ehrenfeld J., Chassot A.A., Ponzio G., Soriani O.

Recent works have indicated that sigma-1 receptor modulates K+ channels. In addition, recent observations suggest that sigma receptors modulate cell proliferation. However, the mechanism by which the sigma-1 receptor inhibit cell proliferation remains enigmatic. In the present study, patch-clamp and western blot assays were used in NCI-H309 (a Small Cell Lung Cancer line) and Jurkat cells (a Leucemic T cell line) to investigate the effects of sigma ligands on K+ channels and cell proliferation. The sigma ligands (+)-pentazocine ((+)Ptz), igmesine (Ig) and ditolylguanidine (DTG) all reversibly inhibited voltage-activated K+ current in both cell lines. The potency of sigma ligands induced-inhibition (10 µM) was Ig (61.7%) = (+)Ptz (57.8%) >> DTG (27.3%), which is in good agreement with the pharmalogical properties of the sigma-1 receptor. Addition of either TEA, a K+ channels blocker, or one of those sigma ligands inhibited proliferative growth of Jurkat as determined by cell counts over 4 days. Furthermore, TEA and sigma ligands caused an accumulation of the cyclin dependent kinase inhibitor p27kip1 but not p21cip1 and a decrease in cyclin A expression in both cell types. This result is consistent with a cell cycle arrest in G1 phase. Several reports suggest that K+ channels are involved in cell proliferation through volume regulation. Interestingly, we found that sigma ligands induced a strong delay in volume regulation when Jurkat cells were challenged with an hypotonic stress. Taken together, these results suggest that the sigma-1 receptor induced modulation of K+ channels may play an important role in cell proliferation arrest in G1 phase through inhibition of cell volume regulation and accumulation of p27kip1.

This work was supported by the Association pour la Recherche sur le Cancer.

UNSA CNRS UMR 6078, Villefranche sur Mer, France

P23-13

CHARACTERIZATION OF THE M-CURRENT IN RAT SENSORY NEURONS

Passmore G.M.(1), Selyanko A.A.(1), Mistry M.(1), Alqatari M.(1), Burbidge S.A.(2), Brown D.A.(1)

Neuronal hyperexcitability is a feature of epilepsy and both inflammatory and neuropathic pain. M-currents (IK(M)) play a key role in regulating neuronal excitability and mutations in neuronal KCNQ2/3 subunits, the molecular correlates of IK(M), have previously been linked to benign familial neonatal epilepsy. In this study, using whole-cell perforated-patch recording, we sought the presence of IK(M) in cultured dorsal root ganglion (DRG) neurons from 17 day old rats. In 30 small cells tested (capacitance: 20.4±1.1pF), of which 16/22 were sensitive to capsaicin, IK(M) was the dominant subthreshold sustained current. IK(M) was also present in the majority (9) of large cells (capacitance: >100pF, n=10) tested, but in contrast to small cells, was masked by large dendrotoxin-sensitive (100nM) and Cs+-sensitive (1mM) currents. As in superior cervical ganglion (SCG) neurons, IK(M) in small DRG neurons activated at ~-60mV and deactivated slowly (tfast=76.4±9.9ms, tslow=583±134ms, n=9). IK(M) was inhibited by the M-channel blocker linopirdine (IC50: 2.1±0.2µM; n=8), its analogue XE991 (IC50: 0.26±0.01µM; n = 6) and Ba2+ (IC50: 0.3±0.04mM; n=4). Sensitivity to TEA ranged from low to intermediate (IC50: 0.2-4.7mM; n=7) indicating a variable expression of TEA-sensitive and -insensitive KCNQ subunits, which was confirmed by immunocytochemistry. As expected, retigabine (10µM) enhanced IK(M) in a voltage-dependent manner (EC50s: 0.18±0.02 µM and 1.19±0.07 µM at –20 and –50mV respectively, n=7). Furthermore, linopirdine (10µM) and retigabine (10µM) reduced and increased the threshold of firing, respectively. RT-PCR confirmed the presence of all four neuronal KCNQ subunits in whole DRG, though KCNQ4 was absent at the single cell level. Thus, in small DRG neurons, native M-channels are composed mostly of either homomeric KCNQ2 or heteromeric KCNQ2/3 subunits.

Supported by the U.K. MRC, the Wellcome trust and EU FP5 programme.

(1)Dept. Pharmacology, UCL, Gower St., London, UNITED KINGDOM

(2)Neurology & GI CEDD, GSK, North Frontiers Science Park, Harlow, UNITED KINGDOM

P23-14

SUBCELLULAR LOCALIZATION OF THE DELAYED RECTIFIER POTASSIUM CHANNELS KCNQ1 AND ERG1 IN THE RAT HEART

Jorgensen N.K., Rasmussen H.B., Moller M., Knaus H.G., Jensen B.S., Olesen S.-P.

Several potassium channels are responsible for the repolarization of the cardiac action potential, these include transient outward and delayed rectifier potassium currents. In the present study the cellular and subcellular localization of the two delayed rectifier potassium channels KCNQ1 and ERG1 was investigated in the rat heart. Confocal immunofluorescence microscopy of atrial and ventricular cells revealed that both KCNQ1 and ERG1-immunoreactivity was confined to the peripheral sarcolemma and to a structure transversing the myocytes. Immunoelectron microscopy of ventricular myocytes showed that the ERG1 channel was selectively expressed in the transverse tubular system and its entrance while KCNQ1 was detected in both the periheral sarcolemma and in the T-tubules. Thus, while ERG1 displays a very restricted subcellular localization pattern, KCNQ1 is more widely distributed within the cardiac cells. The localization of these potassium channels to the transverse tubular system close to the Ca2+ channels renders them with maximal repolarizing effect.

The Panum Institute, University of Copenhagen, DK-2200, Denmark

P23-15

COMPARISON OF AJMALINE- AND PROPAFENONE-INDUCED BLOCK OF ITO IN RAT VENTRICULAR MYOCYTES

Bahnikova M., Matejovic P., Pasek M., Simurdova M., Simurda J.

The effects of ajmaline on the transient outward current (Ito) was studied and compared with those of other class I antiarrhythmic drug propafenone in experiments on rat ventricular myocytes. In our previous work we have shown that the ajmaline-induced block of Ito is frequency-independent in the range of 0.33 - 3.3 Hz. In contrast, propafenone showed significant increase of Ito-block with increasing frequency in the same range. The present study was aimed to explain the observed differences. In experiments on isolated rat ventricular myocytes, Ito was recorded in response to imposed standard depolarizing pulses preceded by variable preconditioning using the whole-cell patch-clamp technique. The reconstructed variations of the degree of block induced by both drugs during a depolarizing pulse increased at first and then decreased in parallel with inactivation. The recovery from the rest of propafenone-induced block after repolarization proceeded slowly (half time around 40 ms). This slow process that is likely to account for the observed frequency dependence of a steady state block was not significant in the case of ajmaline. Consequently, the repeated depolarizations did not induce cumulative block within the explored frequency range. However, both drugs revealed a cumulative block in the range of 20 - 50 Hz if trains of short (10 ms) depolarizing pulses were applied after a 10 s period of rest. The present results show that in the presence of both drugs the inhibition of Ito is mediated mainly through interaction with open channel (Kd = 3.55 mM for propafenone and 3.7 mM for ajmaline). Furthermore, they are consistent with the hypothesis that the apparent affinity of the drug to its receptor is low in the resting and the inactivated state. The differences in the values of rate constants related to recovery from the block of inactivated channels may account for the observed differences in frequency dependence of Ito-block induced by ajmaline and propafenone.

Department of Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic

P23-16

CEREBROVASCULAR DYSFUNCTION IN INSULIN RESISTANT ZUCKER OBESE RATS

Busija D., Erdos B., Miller A.

Our previous in vitro studies indicated that cerebrovascular dilatory responses are reduced in fructose fed insulin resistant (IR) rats. In order to further our understanding of the effect of IR on the cerebral circulation, we examined the vascular function of the basilar artery (BA) using a cranial window preparation in anesthetized Zucker obese (ZO) and lean (ZL) rats. The resting diameters of the BAs were similar in the two experimental groups. Acetylcholine induced endothelium-dependent, NO-mediated dilations in the BAs, but the responses were significantly reduced in the ZO rats. Dilations to acetylcholine were 17±3% (10-6 M) and 26±4% (10-5 M) in ZL and 6±1% and 12±2% in ZO rats (n=6, p<0.01). In contrast, relaxations to sodium nitroprusside were similar in the two experimental groups. Iloprost, a prostacyclin analogue, induced BKCa-channel mediated relaxations in the BAs, however the responses in ZO rats [3±1% (10-7 M) and 10±1% (10-6 M)] were significantly lower compared to the ZL rats [6±1% and 17±1% (n=6, p<0.05)]. Similarly, relaxations to the KATP-channel opener cromakalim were impaired in ZO rats [5±1% (10-6 M) and 28±4% (10-5 M)] compared to ZL rats [17±3% and 43±3% (n=6, p<0.05)]. These findings demonstrate that both endothelium-dependent and smooth muscle K-channel-mediated cerebrovascular responses are severely impaired in IR. Supported by: NIH grants HL-30260, HL-46558, HL-50587 (DWB) and HL-66074, HL-65380 (AWM).

Wake Forest University, Winston-Salem, USA

P23-17

EFFECT OF THE ANTI-CUNVULSANT DRUG, RETIGABINE, ON SINGLE KCNQ2/3 CHANNEL ACTIVITY IN CHO CELLS.

Tatulian L., Brown D.A.

Retigabine is a novel anticonvulsant compound that is now in clinical phase II development. We have previously shown that it enhances whole cell currents generated by KCNQ2/3 K+ channels when expressed in Chinese hamster ovary (CHO) cells, as well as currents through native M channels in rat sympathetic neurones [thought to be generated by KCNQ2/3 subunits]. In the present experiments, we have examined the effect of retigabine on the properties of single heteromeric KCNQ2/3 channels expressed in CHO cells, to see if the increase in macroscopic current amplitude caused by retigabine can be attributed to an increase in the number of functional channels, single channel conductance and/or open probability. KCNQ2/3 potassium channel subunits were co-expressed in CHO cells and currents through single channels recorded using cell-attached patches. Channels had a similar slope conductance in the presence (8.04 + 0.02 pS) and absence (7.6 + 0.01 pS) of 10 μM retigabine. The mean maximal open probability Po for single KCNQ2/3 channels was 0.13 + 0.02, with a half-maximal Po potential (Vo) of -28.7 + 1.4 mV for control recordings. Retigabine increased mean maximal Po to 0.38 + 0.04 and produced a hyperpolarising shift of (Vo) to -40.1 + 3.4 mV. Single KCNQ2/3 channels have multiple voltage-dependent kinetic components in their activity (CL-OS-CM-OL-CS), giving short, medium and long closed times (tauCS, tauCM, tauCL) and short and long open times (tauOS and tauOL). In the presence of retigabine at 0 mV the combined duration and contributions of the longest closed time tauCL decreased ten-fold, while the short and long open times increased four-fold and two-fold respectively. Thus, the results suggest that retigabine enhances macroscopic KCNQ2/3 currents by increasing the channel open probability and modifying steady-state kinetics to favour the open channel configuration.

Supported by the U.K.MRC, the Wellcome Trust and the EU FP5.

Department of Pharmacology, UCL, London, UINTED KINGDOM

P23-18

REGULATION OF CALCIUM-ACTIVATED POTASSIUM CHANNELS BY EXTRACELLULAR ATP

Hede S.E., Amstrup J., Klærke D.A., Novak I., Krogh A.

Extracellular ATP is an important regulator of transepithelial transport in a number of tissues. In pancreatic ducts, results from our laboratory have shown that ATP modulates epithelial K channels via purinergic receptors, most likely of the P2Y2 and P2Y4 types, but so far the identity of the involved K channels is not clear. The aim of the present study was to find which K channels are expressed in pancreatic ducts, and to study regulation of individual K channel types via specific P2Y receptors in an expression system.

RT-PCR experiments showed that Ca-activated K channels of intermediate conductance (IK-channels) and big conductance (BK channels) are abundantly expressed in pancreatic ducts. Possible interactions between the purinergic receptors and the different type of K channels were examined in co-expression experiments in Xenopus laevis oocytes. cDNA for P2Y receptors and K channels were cloned into pXOOM vector. Channel activity was measured electrophysiologically in oocytes stimulated with ATP or UTP (0.1 – 1 mM, pH 7.4). BK channels seemed to be differentially regulated by P2Y2 and P2Y4 receptors. UTP or ATP stimulation of oocytes expressing BK channels and P2Y4 receptors resulted in a 30% increase in the current through the expressed channels (n=6), whereas the BK channels co-expressed with P2Y2 receptors were inhibited by 18-25% during exposure to nucleotides (n=10 for ATP, n=8 for UTP). In contrast, co-expression of IK channels with P2Y4 receptors resulted in a large activation of the current when stimulated by UTP (from 100 to 2000 nA, n=9). This study indicates that there is a differential interaction between the purinergic receptors and different types of Ca-activated K channels.

Institute and Panum Institute, University of Copenhagen, Denmark

P23-19

QT PROLONGING EFFECTS OF DOFETILIDE, A HERG POTASSIUM CHANNEL BLOCKER IN VARIOUS EXPERIMENTAL MODELS

Orosz Sz., Komlódi Zs., Makó É., Farkas S., Dézsi L.

Investigation of the potential QT prolonging effects of new drug candidates is necessary in the early phases of drug research. For this purpose we applied isolated rabbit heart, anaesthetised rabbit and conscious dog models. In the isolated rabbit heart changes in left ventricular pressure (LVP), coronary flow (CF), heart rate (HR) and surface ECG were recorded, and corrected QT (QTc) values were determined. In the anaesthetised rabbit, mean arterial blood pressure (MABP), HR, and QTc derived from standard bipolar ECG were measured. In the conscious dog, HR and QTc were derived from the ECG. In order to validate these models, the effects of dofetilide, a class III antiarrhythmic drug, were investigated.

In the isolated heart LVP and CF were not affected by intracoronary perfusion with dofetilide (10-100 nmol/l), HR dose-dependently decreased from the basal HR (mean BHR: 104 bpm) (to 59 %) whereas QTc dose-dependently increased (to 162 % upon application of 100 nmol/l dofetilide). When the concentration was increased up to 1 μmol/l, the shape of the ECG curve was significantly distorted.

In the anaesthetised rabbit, MABP and HR (BHR: 326 bpm) slightly decreased upon i.v. infusion of increasing cumulative dosing of dofetilide. There was only a slight dose-dependent increase in QTc (to 115 % at 300 μg/kg, i.v.).

In the conscious dog, following oral administration of 0.2 mg/kg dofetilide the HR (BHR: 99 bpm) slightly and transiently decreased (to 90 %). The maximal QTc prolongation (to 116 % of control) was observed at 2 hours post-dose.

In summary, the applied methods are suitable to detect QT prolonging effects. However, in the in vivo models, the degree of QT prolongation that could be achieved with dofetilide was much less pronounced than in the isolated rabbit heart with relatively low heart rates.

Department of General Pharmacology, Gedeon Richter Ltd., Budapest, Hungary

P23-20

KIR6.1 SUBUNIT OF K(ATP) CHANNEL IS PRESENT IN VENTRICULAR CARDIOMYOCYTES OF RABBIT BUT NOT OF RAT

Christe G., Garnier-Raveaud S., Cand F., Usson Y., Verdetti J.

It is widely accepted that the pore of ATP-sensitive K+ (KATP) channels of cardiomyocytes is a tetramer of the Kir6.2 subunit. Here we report on the immunolocalization of Kir6.1 and Kir6.2 KATP channel subunits in ventricular cardiomyocytes. We used antibodies raised to short peptides with specific sequence to each of these subunits (Kir6.2, courtesy of Dr. Grigory Krapivinsky; Kir6.1, courtesy of Dr. Idelson, Alomone Labs, Israel). We verified the selectivity of these antibodies to subunits expressed in Xenopus Oocytes with SUR2A (Courtesy of Dr. Michel VIVAUDOU, CEA Grenoble, France). Ventricular cardiomyocytes were isolated from rabbit and rat hearts, fixed and permeabilized. Primary Abs were applied overnight at 4°C, Alexa 488 labelled secondary antibodies (Molecular Probes) were used to detect primary Abs under confocal microscopy. Kir6.2 was present in both species with a high level of fluorescence in T-tubules, where it colocalized with a-actinin. Kir6.1 labeling was detected in rabbit ventricular myocytes principally at the level of T-tubules, but with lower fluorescence intensity than Kir6.2 labeling. It was undetectable when anti-Kir6.1 antibody was saturated with its specific immunogenic peptide. In rat ventricular myocytes, no Kir6.1 labeling was found. These results point out to possible interspecific differences in Kir6.1 expression. It is still debated whether Kir6.1-Kir6.2 chimeric channels may form in cardiac cells (Seharaseyon et al. J. Biol. Chem. 2000, 275:17561-65; Babenko and Bryan J. Biol. Chem. 2001, 276:49083-92). This issue is important, as such chimeric channels might more readily respond to metabolic depression than canonical Kir6.2 tetrameric channels (Babenko and Bryan 2001). The present finding of both subunits in rabbit ventricular myocytes at the T-tubule membrane suggests more detailed examination of this possibility in native KATP as a possible regulatory mechanism of the metabolic sensing and protection in cardiomyocytes.

Laboratoire de Développement et Vieillissement de l'endothélium INSERM E0219, DRDC / DVE, CEA F-38054 Grenoble, France

P23-21

LARGE CONDUCTANCE K CURRENTS IN HUMAN CORNEAL EPITHELIAL CELLS.

Takahira M., Sakurai M., Sakurada N., Sugiyama K.

Purpose: The large conductance K channel in the corneal epithelium plays critical roles in regulating cell volume and the resting membrane potential. Recently we reported that this K current in the bovine corneal epithelium was also activated by some fatty acids including arachidonic acid. In this study on the human corneal epithelial cells, pharmacological properties of K currents were investigated to identify the channel type.

Methods: Human corneal epithelial cells (HCEC, Kurabo, Japan) were cultured and isolated at the end of passage 4 or 5. The cells were perfused by HEPES-buffered Ringer solutions. Whole-cell currents were recorded using the perforated patch configuration of the patch clamp technique.

Results: The zero potential of HCEC averaged -12 mV (n=78). HCEC expressed a noisy, non-inactivating outward current. No voltage-gated K current was detected. External application of flufenamic acid (0.1 mM, n=18) augmented an outwardly rectifying, noisy current with a reversal potential near the equilibrium potential of K (-80 mV), indicating high selectivity for potassium. Similar K current augmentation was induced by arachidonic acid (0.02 mM, n=10) and by palmitoleic acid (0.1 mM, n=8). The enhanced K current was blocked almost completely by subsequent application of 0.1 mM diltiazem. Gadolinium (0.5 mM) enhanced transiently (< 2 min) the fenamate-induced current augmentation.

Conclusions: The human corneal epithelium exhibited the large conductance K current. The transient current augmentation by gadolinium indicates its dual modulation of the K channel.

Dept of Ophthalmology, Kanazawa University, Kanazawa, Japan.

P23-22

ALLOSTERIC INTERACTION BETWEEN NICORANDIL AND NUCLEOTIDES ON SULFONYLUREA RECEPTORS 2A AND 2B

Yamada M., Kurachi Y.

To understand how nicorandil allosterically interacts with nucleotides on sulfonylurea receptors (SUR) 2A and 2B, we analyzed with the patch clamp method the effect of nicorandil on ATP-sensitive K+ channels formed from Kir6.2 and either SUR2A and SUR2B. SUR2A and SUR2B are respectively cardiac and vascular SUR and differ only in the C-terminal 42 amino acids (C42). An invariant lysine in the Walker A motif in either or both of the nucleotide-binding domain (NBD) 1 and 2 was substituted with alanine (K708A and K1349A, respectively). In the presence of MgATP (1mM), nicorandil activated SUR2A/Kir6.2 and SUR2B/Kir6.2 channels with EC50 of 1.6 mM and 10 µM, respectively. In the presence of MgATP (1mM) and MgADP (100µM), nicorandil activated SUR2A/Kir6.2 channels with EC50 of 80 µM. All these responses were abolished by the K708A or K708A / K1349A mutation. The K1349A mutation did not change the EC50 of nicorandil for SUR2A/Kir6.2 channels in the presence of MgATP alone but increased the EC50 to 2.0 mM in the presence of MgATP and MgADP. This mutation increased the EC50 of nicorandil for SUR2B/Kir6.2 channels to 300µM in the presence of MgATP alone. Therefore, (A) The NBD1-ATP interaction is essential for the effect of nicorandil on both the channels. (B) The effect of nicorandil on SUR2A/Kir6.2 channels is mainly supported by NBD1-ATP interaction alone in the presence of MgATP but also by NBD2-ADP interaction in the presence of MgATP and MgADP, accounting for the more potent effect of nicorandil in the presence than absence of MgADP. (C) The effect of nicorandil on SUR2B/Kir6.2 channels is supported by both NBD1-ATP and NBD2-ATP interactions in the presence of MgATP. Furthermore, the NBD1-ATP interaction more strongly supports the effect of nicorandil on SUR2B than SUR2A. Thus, nicorandil much more potently activates SUR2B/Kir6.2 than SUR2A/Kir6.2 channels. (D) The C42 likely modulates allosteric interaction between nicorandil-binding sites and NBDs on SUR2A and SUR2B.

Department of Pharmacology II, Graduate School of Medicine Osaka University

P23-23

PHYSIOLOGICAL SIGNIFICANCE IN ALLOSTERIC MODULATION OF RGS PROTEINS BY PIP3 AND CALMODULIN

Ishii M., Kurachi Y.

Regulators of G-protein-signalling (RGS) proteins are a family of proteins, which accelerate intrinsic GTP-hydrolysis on a subunits of trimeric G-proteins and play crucial roles in the physiological regulation of G-protein mediated cell signaling. If RGS proteins were active unrestrictedly, it would completely suppress various G protein-mediated signallings as has been seen in the over-expression experiments of RGS proteins. Therefore, it is quite important to understand how the actions of RGS proteins are regulated in various physiological conditions. The modulatory mechanisms of RGS-action per se have, however, been poorly clarified. We recently showed in cardiac myocytes a physiological mode of action of a RGS protein (Circ Res 2001; PNAS 2002). The voltage-dependent formation of Ca2+/calmodulin (CaM) facilitated the GTPase-activity of RGS protein via removing intrinsic inhibition mediated by a kind of phospholipid, phosphatidylinositol-3,4,5,-trisphosphate (PIP3). This modulation of RGS-action underlies the "relaxation" behavior of G-protein-gated K+ (KG) channels in native cardiac myocytes. In order to elucidate their molecular mechanisms, we have performed further examination using co-sedimentation assay, which enables us to perform quantitative analyses on protein-lipid interaction. In results, we detected the specific interaction between RGS4 and PIP3 (but not other PIPs), which was abolished by Ca2+/CaM. Interestingly, the allosteric modulation is exclusively performed within RGS domain, which is also responsible for GTPase-accelerating activity. We identified the clusters of positively charged residues (K99, K100) in helix 4 of RGS domain as a candidate of the switch of PIP3/CaM-modulation. Because the residues are conserved in almost all RGS protein subtypes, the allosteric modulation of RGS proteins should be important in the physiological regulation of G-protein signalling by various RGS proteins in different cell types.

Department of Pharmacology II, Graduate School of Medicine, Osaka University, Suita, JAPAN

P23-24

BIOCHEMICAL EVIDENCE FOR THE INTERACTION OF THE HSK3 CHANNEL WITH A PROTEIN OF THE ENDOPHILIN FAMILY

Hoppner A.C., Spindler I., Wälter S., Wanker E., Lehmann-Horn F., Grissmer S., Jäger H.

Small Ca2+ activated K+ channels (SK) generate the slow afterhyperpolarisation (sAHP) following an action potential. The human SK3 channels (hSK3) are widely expressed, in liver, heart, skeletal and smooth muscle, and in the brain. For understanding the physiological role of hSK3 it is important to know modulating factors, protein targeting mechanisms, and localization. To find putative interaction partners we performed a screening for interacting proteins with a LexA-based yeast two-hybrid system. Recently we could show the interaction of the first 299 amino acids of hSK3 with SH4GL3, a member of the endophilin family, while other SH4 domain containing proteins like Abl, Lck, Fyn, Grb2, PI3K P85 did not interact.

To further confirm the interaction in vitro we performed a pulldown assay whereby the coding region of the N-tail of hSK3 was cloned into a prokaryotic expression vector fused to glutathione-S-transferase (GST) and the SH4GL3 gene into a prokaryotic expression vector fused to a His-tag. Both proteins were expressed in E. coli. Expression of both fusion proteins was confirmed by Western blotting. For the pulldown assay the ProFound Pull-down PolyHis Protein:Protein Interacion Kit was used following the manufacturers? instructions. Analysis of the experiment was done by Western blotting with antibodies against GST and SH4GL3. Our data indicate an interaction between hSK3 N-tail and SH4GL3 and hence confirm our previous results found with the yeast two-hybrid system. As SH4GL3 and hSK3 are both expressed in brain nerve terminals the interaction might be possible under physiological conditions and be relevant for channel function modulation or targeting of hSK3.

Supported by grants from the DFG (Gr848/8-2), the BMBF (iZKF Ulm, B7), and the Graduate College 460 "Diagnostic and Therapeutic Concepts in Molecular Medicin?.

Dept. of Applied Physiology, University Ulm, Germany

S24 Pulmonary circulation : from cell to integrative physiology

ORAL SESSION

S24-1

CALCIUM OSCILLATIONS IN PULMONARY SMOOTH MUSCLE CELLS

Marthan R.

Using microspectrofluorimetry to measure the [Ca2+]i in single cells, studies from our laboratory and others have revealed that agonists controlling smooth muscle tone via activation of membrane receptors, such as angiotensin II, endothelin-1, noradrenaline, serotonin induce a complex temporal [Ca2+]i response in pulmonary artery smooth muscle cells (PASMCs). The agonist-induced [Ca2+]i response is composed of a series of cyclic increases in the [Ca2+]i, so-called Ca2+ oscillations. The average percentage of oscillating cells is 50 to 80 % under identical experimental conditions. The concentration of agonist is the main factor that modulates the pattern of Ca2+ oscillations. In contrast to mediators acting at cell surface receptor, caffeine and ryanodine, known to act directly on the sarcoplamic reticulum (SR) always induce a transient or monotonic increase of [Ca2+]i that is never followed by oscillations. The amplitude of this transient [Ca2+]i -response is dependent on the concentration of caffeine. Therfore, agonist-induced Ca2+ oscillations appear to be underlain by a cytosolic Ca2+ oscillator. Both acute and chronic hypoxia (CH) alter resting [Ca2+]i value and Ca2+ transients in PASMCs. The molecular mechanisms of such alterations are not fully understood. We have investigated the effect of CH chronic on calcium signaling in PASMCs. In myocytes from CH rats, the [Ca2+]i response to agonists is altered with a disappearance of Ca oscillations and a decrease in the percentage of responding cells. However, both the amount of mobilized calcium and the sources of calcium implicated in the agonist-induced response are not changed. In conclusion, alterations in pulmonary vascular reactivity are related to hypoxia-mediated effects on various pathways implicated in PASMC calcium homeostasis. Further studies are required to identify the common mechanism(s) leading to these various effects in order to define new molecular therapeutic targets.

Inserm & Université Bordeaux 2, Bordeaux, France

S24-2

NON VOLTAGE-DEPENDENT CALCIUM INFLUX IN AN INTEGRATED MODEL OF PULMONARY MICROVESSELS

Guibert C., Marthan R., Savineau J.-P.

5-hydroxytryptamine (5-HT) is a potent pulmonary vasoconstrictor and contributes to hypoxic pulmonary vasoconstriction and pulmonary arterial hypertension. Small resistance pulmonary arteries are very important for blood flow regulation and very sensitive to hypoxia in the pulmonary vascular bed. Thus, we further investigated the mechanisms involved in the calcium signal to 5-HT in rat small intrapulmonary artery (IPA).

Effects of 5-HT were examined in isolated IPA (external diameter < 250 µm) from rat. Digital imaging with fura-PE3 was used to record intracellular calcium concentration and to follow external diameter of the vessels. Nerves and endothelium were detected by labeling the vessels with anti-neurofilament and anti-endothelial-nitric oxide synthase antibodies respectively.

Tetrodotoxin 0.5 µM and L-NAME 300 µM did not affect the concentration-dependent calcium increase induced by 5-HT. 5-HT 10 µM induced a sustained calcium variation which was sensitive to the inhibitor of the 5-HT2A receptors, ketanserin 0.1 µM, and insensitive to voltage-dependent L-type calcium channel blockers (nitrendipine and nicardipine 1 µM) or voltage-independent calcium channels antagonists (LOE 908, SKF 96365 and gadolinium 10 µM). The calcium response to 5-HT was also not modified by a sarcoplasmic reticulum Ca2+-ATPase inhibitor (cyclopiazonic acid, CPA, 10 µM) which depletes intracellular calcium store. CPA alone activated a capacitative calcium channel which was sensitive to LOE 908 and insensitive to SKF 96365 and gadolinium. The sustained calcium signal to 5-HT was partly blocked by inhibitors of arachidonic acid production (RHC 80267 50 µM and isotetrandrine 10 µM) and mimicked by application of exogenous arachidonic acid.

These results suggest that 5-HT-induced sustained calcium increase in small IPA is partly due to activation of a non-capacitative arachidonic acid sensitive receptor-operated calcium channel.

Laboratoire de Physiologie Cellulaire Respiratoire - INSERM E0356 - Université Bordeaux 2 - Bordeaux - France

OC24-1

SILDENAFIL PREVENTS CHANGE IN RHOA EXPRESSION INDUCED BY CHRONIC HYPOXIA IN RAT PULMONARY ARTERY

Sauzeau V., Rolli-Derkinderen M., Loirand G., Pacaud P.

Exposure to chronic hypoxia (CH) induces a sustained pulmonary hypertension associated with structural and functional changes in the pulmonary arterial bed, including alterations of contractile properties. The small G-protein RhoA and its effector Rho kinase constitute major components of the sustained rise in tension induced by vasoconstrictors. The aim of this study is to analyze the effect of CH on RhoA/Rho kinase signalling pathway in the rat pulmonary artery (PA).

CH (10% O2, 2 weeks) induces a decrease in NO production/disponibility in rat PA, remodelling of PA and right ventricular hypertrophy. Maximal contraction of PA rings to endothelin-1, noradrenaline and the thromboxan A2 analogue U46619 is decreased by 27.7±2.8% (n=4), 45.2±7.0% (n=8) and 73.8±5.4% (n=4) in CH rats respectively. Contraction measurement in permeabilized strips of PA demonstrates that the CH-induced decrease in the response to agonists is due to the abolition of RhoA-mediated Ca2+ sensitization of the contraction. Real-time RT-PCR and western blot analysis reveal that CH induces decrease in RhoA mRNA (79.4±6.0%, n=4) and RhoA expression (81.1±8.0%, n=4) in the main pulmonary artery. Treatment of rats with the type 5 phosphodiesterase inhibitor, sildenafil (25 mg/kg/d) throughout 2 weeks of exposure to CH prevents CH-induced down-regulation of RhoA, reduction of contraction and PA remodelling.

These findings indicate that CH induced-down-regulation of RhoA expression, leading to the abolition of RhoA/Rho kinase-mediated Ca2+ sensitization of the contraction, is responsible for the decreased responses to contracting agonists in pulmonary artery of CH rats. These alterations are prevented by sildenafil, indicating a major role of NO/cGMP pathway in the CH-induced alteration of RhoA signalling in pulmonary artery.

Inserm U-533, Nantes, France

OC24-2

REGULATION OF T LYMPHOCYTE SURVIVAL BY RESIDENT CELLS IN ASTHMA

Darveau M.-E., Rouabhia M., Pagé N., Chakir J.

Background : Asthma is a chronic inflammatory disease where bronchial mucosa is infiltrated by activated T cell. Understanding the mechanisms that govern cell-apoptosis and factors involved may help to explain different aspects of this disease. Signals from tissue microenvironments and resident cells may be a potential mechanism involved in the unrelenting and survival of activated T cells. Objective: This study was designed to investigate the effect of resident cells on survival of T lymphocytes. Methods: We used a well designed engineered human bronchial mucosa (EHBM). Different tissues were produced : an EHBM containing both epithelial cells and fibroblasts, an EHBM with only fibroblasts included in the collagen gel, an EHBM with epithelial cells seeded onto the collagen gel without fibroblasts and T cells cultured in the gel. We performed immunofluorescence to determine the expression of CD45Ro, Bcl-2 and Bax. We also performed Tunel techniques to evaluate apoptosis of T cell cultured with EHBM. Results: We obtained a significant decrease of apoptotic T cells, when they were cocultured with fibroblasts alone (20% ± 6 compared to 32% ± 8), epithelial cells alone (15% ± 6 compared to 32% ± 8) or the two type of resident cells (20% ± 7 compared to 32% ± 8 ) obtained from asthmatics. Fibroblasts and epithelial cells from normal subjects showed no protective effect. The decrease of apoptosis in asthmatic cells was associated with a significant down-regulation (p =0.001) in Bax expression by T cells CD3+ when they were cultured with EHBM from asthmatics compared to T cells cultured in collagen gel. Conclusion: These results support the fact that bronchial resident cells can play a role in the amplification of inflammation in asthma by increasing the survival of T lymphocytes

(supported by CIHR and Canadian Asthma Society).

Centre de recherche de l'Hôpital Laval, Université Laval – Québec – Canada

OC24-3

MODULATION OF HUMAN PULMONARY VASCULAR TONE BY GUANOSINE 3'-5' CYCLIC MONOPHOSPHATE

Perrotin C., Pham-Minh H., Regnard J.F., Dall'Ava-Santucci J., Dinh-Xuan A.T.

Background. Guanosine 3'-5' cyclic monophosphate (cGMP) is a nucleotide second messenger that plays a key role in the mechanisms leading to vasodilatation in response to endothelium-derived nitric oxide (NO). It is possible to increase intracellular concentration of cGMP through stimulation of soluble guanylyl cyclase (sGC) by NO and its donors. Alternatively, it is also possible to keep a high level of intracellular cGMP through inhibition of type 5 isozyme of phosphodiesterase (PDE-5). The aim of our study was to assess the role of cGMP in the modulation of human pulmonary vascular tone and the effects of substances activating sGS and/or inhibiting PDE-5 in human pulmonary vascular smooth muscle.

Methods. Fourth generation pulmonary arteries (PA) were dissected from lungs obtained from 15 patients (3 women and 12 men, age range: 47-79 yrs) undergoing lobectomy for lung carcinoma. Pulmonary vascular rings were mounted in organ chambers and isometric tension was measured in responses to acetylcholine and various agonists stimulating sGC and antagonists inhibiting PDE-5.

Results. Sodium nitroprusside, a NO donor, induced a dose-dependent and maximally relaxed all PA vascular rings, an effect which was significantly greater than that observed with the endothelium-dependent vasodilator, acetylcholine. Relaxation of PA vascular rings was weaker in response to YC-1, a specific activator of sGS, as compared with that observed with zaprinast, a specific inhibitor of PDE-5.

Conclusion. Results from this study are consistent with a critical role of cGMP in the modulation of pulmonary vascular tone. Circumstantial evidence also suggests that sGS and PDE-5, two key enzymes that control cGMP intracellular concentration, are differentially regulated in human pulmonary vascular smooth muscle. Activation of the former and/or inhibition of the latter might be of therapeutical interest in human pulmonary vascular disease.

Laboratoire de Physiologie, CHU Cochin-Port-Royal & Service de Chirurgie Thoracique, Hôpital Hôtel-Dieu, Paris - FRANCE

OC24-4

REN-2 TRANSGENIC RATS HAVE REDUCED CHRONIC HYPOXIC PULMONARY HYPERTENSION

Hampl V., Bíbová J., Herget J., Charles X.

Although there are indications that the rennin-angiotensin axis may participate in the regulation of the pulmonary circulation, its exact role is insufficiently characterized. We used transgenic rats harboring mouse Ren-2 renin gene to characterize its involvement in pulmonary vascular regulation under normal conditions and during chronic hypoxic pulmonary hypertension. Since homozygous rats rapidly develop heart failure, heterozygote (+/-) males were used in this study and compared to wild-type controls (-/-).

First, pulmonary artery pressure (PAP) was measured by catheterization in closed-chest, thiopental-anesthetized rats aged 50 and 80 days. Cardiac output (CO) was estimated as the ascending aorta blood flow by transonic flowmeter. While the systemic blood pressure (measured in anesthesia by carotid artery cannulation) was elevated in +/- rats at 50 days (139±4 mmHg) and 80 days (151±3 mmHg) compared to -/- rats (109±3 and 111±5 mmHg), PAP did not differ between the +/- and -/- rats at 50 days (17±1 vs. 16±1 mmHg) and at 80 days (19±2 vs. 18±1 mmHg). The groups also did not differ in CO at either age. In the next phase of the experiment, rats were exposed to hypoxia (10% O2) for 2 weeks until they were 80 days old. As expected, chronic hypoxia elicited pulmonary hypertension in -/- rats: their PAP was 30±2 mmHg (compared to 18±1 mmHg in normoxic -/- controls; P>0.0001). In +/- rats, chronic hypoxia also elevated PAP (25±2 mmHg), but significantly less than in -/- rats (P>0.05). These differences were not due to differences in CO.

We conclude that chronic hypoxic pulmonary hypertension is milder in the Ren-2 transgenic heterozygotes, while their PAP in normoxia does not differ from -/- controls. Thus, the rennin gene is involved in the development of chronic hypoxic pulmonary hypertension. The Ren-2 transgenic rats are a useful model for further study of this phenomenon.

Laboratoire de Physiologie, CHU Cochin-Port-Royal & Service de Chirurgie Thoracique, Hôpital Hôtel-Dieu, Paris - FRANCE

S24-3

ROLE OF SEROTONIN AND ITS TRANSPORTER IN PATHOGENESIS OF PULMONARY ARTERIAL HYPERTENSION

Adnot S., Eddahibi S.

Recent years have witnessed the identification of biological processes pivotal to the complex vascular changes associated with various forms of pulmonary hypertenion (PH). We recently reported that serotonin (5-hydroxytryptamine, 5-HT) and its transporter (5-HTT) play a critical role in the pulmonary arterial smooth muscle (PA-SMCs) hyperplasia and vascular remodeling associated with experimental hypoxic PH and human primary PH. Serotonin has a potent mitogenic effect on PA-SMCs and this effect is mediated by 5-HTT, not by serotonin receptors. In the experimental model of hypoxic pulmonary hypertension, 5-HTT expression is increased as a result of a direct effect of hypoxia on transcription of the gene. Mice that are deficient in 5-HTT are protected against the development of hypoxic pulmonary hypertension, whereas mice with 5-HTT overexpression develop more severe pulmonary hypertension that do wild-type mice. 5-HTT expression is increased in PA-SMCs and platelets from patients with PH as compared to controls. This 5-HTT overexpression in SMCs from patients persists when the cells are cultured. It is related in part to a functional polymorphism located on the 5-HTT gene promoter: thus, homozygosity for the (L) allele, the long gene promoter variant associated with a high level of gene transcription, is found in 65-75% of patients with primary PH as compared to only 25-30% of controls. It is likely that 5-HTT gene polymorphism, in combination with other factors, may play a crucial role in various forms of pulmonary hypertension. Recent data obtained in patients with advanced hypoxemic lung disease are consistent with this hypothesis.

INSERM U492 and Departement de Physiologie, Hopital H Mondor, 94010, Créteil, France

S24-4

COUPLING BETWEEN THE PULMONARY CIRCULATION AND THE RIGHT VENTRICLE

Naeije R.

Pulmonary hypertension is usually evaluated by mean pulmonary artery pressure, left atrial pressure, cardiac output measurements, and derived pulmonary vascular resistance calculations. While this approach has shown to be useful to define the resistive properties of the pulmonary vascular bed, it is insufficient for the quantification of right ventricular afterload. Ventricular function is optimally defined by a pressure-volume loop, but this approach has been limited until now by the technical difficulties of the continuous measurement of RV volume. We measured in intact anesthetized dogs instantaneous RV pressure and pulmonary arterial flow, and calculated an isovolumic beat maximal RV pressure (Pmax) from non-linear extrapolations of the isovolumic portions of the instantaneous RV pressure curve. Predicted Pmax was closely correlated to maximum RV pressure measurements obtained by direct pulmonary arterial clamping. End-systolic elastance (Ees) of the RV was calculated by drawing a straight line from the Pmax-end diastolic volume point tangential to the systolic portion of the RV pressure-volume curve. Effective pulmonary arterial elastance (Ea) was calculated by drawing a straight line from the Ees point to the end-diastolic volume point. Thus, we were able to obtain a load-independent measurement of RV contractility and an integrated estimate of the forces that oppose RV ejection, and most importantly, the coupling of both. Dobutamine increased and propranolol decreased Ees and the Ees/Ea ratio. We applied this approach to a model of congenital cardiac shunt-associated pulmonary arterial hypertension induced by three months systemic-to-pulmonary shunting in growing piglets. In this model, both RV Ees and pulmonary Ea were increased, with unaltered coupling. Chronic oral bosentan therapy, or acute iv prostacyclin, or inhaled NO had no intrinsic effect on RV contractility.

Free University of Brussels, Brussels, Belgium

S24-5

USING TRANSGENIC MICE TO UNDERSTAND MECHANISMS OF PULMONARY HYPERTENSION

Rodman D.M., Fagan K., West J.

Purpose: Utilize genetically engineered mice to understand mechanisms underlying pulmonary vascular disease.

Methods: Hemodynamic phenotyping of transgenic mice was performed using two techniques: 1) Measurement of PA systolic pressure via trans-thoracic right ventricular puncture and 2) isolated perfused mouse lung. Morphometrics were also performed on fixed tissue. The following transgenic mice constructed by others were used: nitric oxide synthase null mice, pre-pro-ET1 transgenic mice, prostacycline synthase transgenic mice. The following transgenic mice were constructed by us: dominant-negative bone morphogenetic protein receptor-II (BMPRII) transgenics. The latter utilized a tissue-specific conditional approach using double transgenic mice.

Findings: 1) The role of endothelial vasodilator control of pulmonary vascular tone and structure was investigated in NOS null and PGIS transgenic mice. NOS3 was identified as the principle isoform modulating pulmonary vascular tone in the lung, although extrapulmonary NO production, likely from upper airway NOS2, contributed as well. Pulmonary specific overexpression of PGIS markedly reduced the development of hypoxic pulmonary hypertension in PGIS transgenic mice, suggesting that endogenous PGIS activity is rate limiting. 2) The role of ET-1 in the pathogenesis of pulmonary vascular disease was investigated in ET1 transgenic mice. A remarkable lymphocyte-predominant fibrotic vasculitis was seen. 3) dnBMPRII mice were constructed to determine the mechanisms through which loss of BMPRII results in familial pulmonary hypertension. Preliminary phenotyping of these mice will be presented.

Conclusions: Transgenic mice have proven to be a flexible and useful tool in dissecting he mechanisms underlying the pathogenesis of pulmonary vascular disease. Utilizing newer technology we have constructed conditional tissue-specific dnBMPRII transgenic mice. Phenotyping is in progress.

Center for Genetic Lung Disease, University of Colorado, Denver, CO, USA

POSTER SESSION

P24-01

HEART RATE AND MUSCLE PHOSPHATE METABOLISM PROVOKED BY BREATH-HOLDING AND SIMULATED DIVING IN MAN

Rasmussen T.C., Paulev P.-E., Quistorff B.

Face immersion in cold water is a strong stimulus for diving bradycardia and a widely used technique for simulated diving. The object of the present investigation was to measure the heart rate reduction and the phosphate metabolism in the antebrachial flexor muscles during breath-holding and during simulated diving in cold water, during a standardized handgrip manoeuvre.

The investigation was carried out on 7 healthy male volunteers including one of the authors. Handgrip manoeuvres were performed with the left arm in the nuclear magnetic resonance spectrometer and quantified using a plast tensiometer. The heart rate was counted using a plast stethoscope.

We found a relative bradycardia during breath holding at room air (up to 25%). During apnoea with the face immersed in cold water the heart rate was reduced up to 50%.

Evolutionary hypoxia adaptation implies that anaerobic pathways are favoured that maximize the generation of ATP per mol of oxygen. We found no sign of such an adaptation in the human forearm muscles neither during breath holding nor during face immersion in cold water. Oxygen sensitive tissues such as the brain and the myocardium may react differently.

Department of Medical Physiology & Nuclear Magnetic Resonance Center, The Panum Institute, University of Copenhagen - Denmark

P24-02

MODULATION OF PULMONARY VASCULAR TONE BY THE SPECIFIC PHOSPHODIESTERASE TYPE 5 INHIBITOR SILDENAFIL.

Savineau J.P., Pauvert O., Lugnier C., Keravis Th., Marthan R., Rousseau E.

Cyclic nucleotides (cAMP and cGMP) are involved in the control of pulmonary vascular tone. Cyclic nucleotide level is controlled by specific phosphodiesterases (PDE) which are the only enzymes hydrolysing cyclic nucleotides. Recently, drugs acting on specific cGMP PDE (type 5) have been shown to have a potent pulmonary relaxant effect. In the present work we have investigated the presence of PDE5 isozyme and the effect of sildenafil (viagra), a potent PDE5 inhibitor, on the specific cyclic nucleotide phosphodiesterase (PDE) activity, smooth muscle tone and calcium signaling in the rat main pulmonary artery (MPA).The PDE activity was measured in cytosolic and microsomal fractions. Total cAMP and cGMP-PDE activities were mainly present in the cytosolic fraction. Sildenafil (0.1 µM) reduced by 72% cGMP-PDE activity whereas zaprinast (10 µM), a relatively selective PDE5 inhibitor, reduced this activity by 63%. Western blot analysis revealed the expression of PDE5 mainly in the cytosolic fraction of MPA. Sildenafil concentration-dependently inhibited (IC50 = 3.4 nM) the activity of MPA PDE5 partially purified by HPLC. Sildenafil (0.1 nM-50 µM) concentration-dependently relaxed MPA rings pre-contracted with phenylephrine (0.5 µM). The potency of sildenafil (IC50 = 11 nM) was similar to that of a nitric oxide donor, sodium nitroprusside, but higher than that of zaprinast (IC50 = 600 nM). In isolated MPA myocytes, which had been loaded with the calcium fluorophore indo-1, sildenafil (10-100 nM) antagonized ATP- and endothelin-1- induced calcium oscillations but had no effect on the transient caffeine-induced [Ca2+]i-response. This study demonstrates the presence of a functional and highly sildenafil-sensitive PDE5 isozyme in rat MPA. Inhibition of this isozyme mainly accounts for the potent pulmonary vasodilator action of sildenafil which involves alteration in the inositol triphosphate-mediated calcium signaling pathway.

Laboratoire de Physiologie Cellulaire Respiratoire, INSERM (0356), Université Bordeaux 2, Bordeaux, 33076, France.

P24-03

PULMONARY HEMODYNAMICS DURING AN INTERMITTENT EXERCISE TEST IN COPD PATIENTS

Lonsodrfer-Wolf E., Bougault V., Richard R., Oswald-Mammosser M.

The purpose of our study was to evaluate the behavior of the pulmonary arterial pressure (PAP) during a 30 minute intermittent exercise test (IET) in patients with chronic pulmonary obstructive disease (COPD).

Methods: Seven COPD patients, (six males, one women, 58 ± 5 years), with a FEV1 of 47 ± 11% of predicted, and seven healthy volunteers (38 ± 5 years) underwent a maximal exercise test (MET) in the morning and an IET in the afternoon of a same day, with a catheter inserted in the pulmonary artery. The 30 minute IET consisted of six stages alternating four minutes of work set on the first ventilatory threshold determined during the MET, with one minute work set on 90% of the maximal tolerated power of the MET, which corresponded in our series, to the second ventilatory threshold for the healthy subjects. Ventilatory parameters, mean pulmonary artery pressure (PAP), cardiac output (CO) using the Fick’s method, heart rate (HR) were measured, and stroke volume (SV) was calculated. Results: PAP increased during the first minutes of the IET (without reaching the maximal pressure measured at the end of the IET), with a further subsequent significant decrease until the end of the test. CO increased and then remained stable, HR increased regularly reaching the maximal value of the MET at the end of the 30 minute IET. After an initial increase, SV decreased significantly throughout the IET. Total pulmonary vascular resistance (TPVR) decreased in COPD patients, but less than in healthy subjects. Conclusion: i) Despite bouts of hard work, there is no dramatic increase in PAP during the IET in our patients. ii) When comparing the behavior of PAP during the IET in COPD to that observed in younger normal subjects, there was no difference in the slope of decrease of PAP, although PAP was consistently higher in COPD than in normal subjects. iii) TPVR decreased in COPD patients but to a lesser extent than in healthy but also younger subjects.

Explorations Fonctionnelles Respiratoires et de l’Exercice -HUS - 67091 Strasbourg Cedex - France

P24-04

SURFACTANT LUNG LAVAGE AND HIGH-FREQUENCY JET VENTILATION (HFJV) IN EXPERIMENTAL MECONIUM ASPIRATION

Sevecova D., Calkovska A., Drgova A., Javorka M., Javorka K.

Background: Combined therapeutic approach is needed to overcome respiratory failure due to meconium aspiration syndrome (MAS) in newborns. Effects of surfactant lung lavage and HFJV on gas exchange, dynamic lung-thorax compliance (Cdyn), right-to-left pulmonary shunts (RLS) and removal of meconium were evaluated in a rabbit model of MAS. Methods: Suspension of human meconium (25 mg/ml, 4 ml/kg) was instilled into the tracheal cannula of conventionally ventilated (CV, f. 30/min) adult rabbits. When respiratory failure developed, lung lavage (10 ml/kg in 3 portions) was performed either with diluted surfactant (Curosurf, 100 mg phospholipids/kg) or saline during CV or asymmetric HFJV (f.300/min, inspiration time Ti 70%). After the lavage, animals were further ventilated for 1 hour either with CV or HFJV (Ti 50%). Blood gases and lung function parameters were evaluated before and after meconium administration and 10, 30 and 60 min after the lavage. Removal of meconium was estimated spectrophotometrically and by mecocrit method. One-way ANOVA with LSD post-hoc test was used for between-group comparisons, considered p<0.05 for statistically significant. Results: Improvement in oxygenation index after the lavage was observed in both surfactant groups (p<0.05), although was slightly pronounced in conventionally ventilated animals of Surf-CV group. Higher elimination of CO2 after the lavage was found in Surf-HFJV group vs. all remaining groups (p<0.05). Cdyn increased and RLS decreased after the surfactant lavage in both surfactant groups compared to saline controls (p<0.05). Removal of meconium was higher in surfactant groups than in controls (p<0.05) and also higher in Surf-HFJV vs. Surf-CV (p<0.05). Conclusion: Surfactant lung lavage with HFJV improved gas exchange, increased lung compliance and reduced pulmonary shunting by the similar manner as surfactant lavage with CV, and in addition, increased the removal of meconium.

Dept. of Physiology and Dept. of Biochemistry, Comenius University, Jessenius Faculty of Medicine, Martin, Slovakia

P24-05

ACCURACY OF 2-KM WALK TEST PREDICTED VO2MAX IN ACTIVE SENIORS

Rance M., Lazaar N., Boussuge P-Y., Bedu M., Van Praagh E., Duché P.

Direct measurement of maximal oxygen consumption (VO2max) requires maximal effort of the subject and substantial laboratory readiness. In large, middle-aged and elderly populations, this direct time consuming method is inappropriate. The 2-km Walk Test is a simple test, which offers VO2max prediction equations for working age population. Purpose: To explore the accuracy of VO2max prediction equations of the 2-km Walk Test in active seniors aged 60-76. Methods: Twenty-seven active women and men (67±4 yr) participated in the study. VO2peak (l/min) was measured during cycle ergometry by direct gas analysis from a maximal test (step: 30 Watts, time: 2min30). VO2peak related to body mass (BM) was then calculated (ml/min/kgBM). Subjects completed also the 2-km Walk Test (UKK INSTITUTE). VO2max (l/min; ml/min/kgBM) was then predicted from age, sex, Body Mass Index, heart rate and walking time measured during the 2-km Walk Test. Relationships between predicted VO2max and measured VO2peak values were observed calculating correlation coefficients. Measured values were compared with 2-km Walk Test prediction equations via mean difference analyses, and bias was explored using Bland-Altman analyses. Results: Predicted VO2max and measured VO2peak were high correlated (r>0.72, p<0.001). Predicted VO2max (1.33±0.5 l/min; 22±6.8 ml/min/kgBM) was not significantly different from measured VO2peak (1.37±0.49 l/min; 20.19±4.8 ml/min/kgBM). By using Bland-Altman plots, predicted VO2max showed no significant bias. Conclusion: The 2-km Walk Test offers an accurate VO2max prediction in an active senior population though a pretty important bias could be observed in few subjects. This test can be used to evaluate VO2max in a population presented these characteristics.

Lab. BAPS, University Blaise Pascal and University d’Auvergne, Clermont-Ferrand, France

P24-06

INHALED NITRIC OXIDE IMPROVES HEMODYNAMIC CHANGES FOLLOWING ACUTE MASSIVE CO2 PLUS 10% N2O PULMONARY

Mobacher K., Diemunsch P., Doutreleau S., Lupescu R., Schaefer A., Plobner P., Mettauer B., Piquard F., Geny B.

Background : Nitrous oxide (N2O), used during laparoscopic anesthesia, accumulates in CO2 pneumoperitoneum (10% of N2O is often observed) and gazeous embolism is a rare but life-threatening complication.

Objectives : To determine the pulmonary deleterious effects of an acute CO2 + 10% N2O embolism in experimental pigs and to investigate the potential beneficial effect on inhaled NO in such setting.

Methods : Seven anesthetized, ventilated and instrumented pigs underwent three randomly designed gazeous pulmonary embolisms (2cc/Kg during 1 minute) through the jugular vein without or with either inhaled NO at 7 and 22 ppm.

Results : CO2 + 10% N2O increased heart rate (from 82.4±8.4 to 92.3±9.4 bpm, P = 0.02), mean pulmonary artery pressure (from 16.9±09 to 29.9±2.3 mm Hg, P<0.002), systemic vascular resistances (from 1.25± 0.12 to 1.60±0.12 dynes.cm-5.s, P=0.04), pulmonary vascular resistances (from 0.36± 0.03 to 0.78±0.15 dynes.cm-5.s, P=0.02) and central venous pressure (from 2.6±0.6 to 3.0±0.7 mm Hg, P<0.005). It decreased mean arterial pressure (from 57.5±3.4 to 50.8±2.3 mm Hg, P=0.02), end CO2 expiratory pressure (from 36.0±1.8 to 23.7±2.0 mm Hg, P<0.01) and cardiac output (from 3.5±0.5 to 3.2±0.4 L.min-1, P=0.02). NO inhalation attenuated pulmonary hypertension by 34±8 %, P<0.05, and PetCO2 decrease was reduced by 31± 7%, P<0.05.

Conclusion : Thus, inhaled NO, a selective pulmonary vasodilatator, protects against CO2 + 10% N2O gazeous-induced pulmonary hypertension. The dose of 7 ppm might therefore be of therapeutic value during surgical laparoscopy. There is no need to increase the dose of inhaled NO to 22 ppm.

Physiology and Anesthesiology Departments. EA 3072, Faculté de Médecine et HUS, 67000 Strasbourg, France

P24-07

EFFECT OF PHOSPHODIESTERASE INHIBITION ON PULMONARY HYPERTENSION IN CHRONIC HYPOXIC RATS

Andersen C.U., Mulvany M.J., Simonsen U.

Pulmonary hypertension (PH) is characterized by vasoconstriction and vascular remodelling. In smooth muscle cells, cGMP is a vasodilating messenger. Nitric oxide (NO) stimulates the guanylyl cyclase (GC) to produce cGMP, which is hydrolysed by the phosphodiesterase 5 (PDE5). This study was designed to investigate the effect of the NO-donor, molsidomine, and the PDE5 inhibitor, sildenafil, in pulmonary arteries and as treatments for PH in chronic hypoxic rats. Rat pulmonary arteries were dissected and mounted in wire myographs. Concentration-response curves (CRCs) for sildenafil and the active metabolite of molsidomine, SIN-1, were performed in endothelium-intact arteries in the absence and presence of an inhibitor of soluble GC, ODQ (3 uM). The rats were divided in 5 groups: A normoxic and a hypoxic control group, and three hypoxic groups receiving either sildenafil, molsidomine, or the combination. After two weeks, systemic blood pressure, right ventricular pressure (RVP), and right ventricle to left ventricle plus septum ratio (RV/LV+S) were measured. Sildenafil evoked maximal relaxations of 40±7% (n=10) in isolated endothelium-intact arteries. Sildenafil relaxation was inhibited by endothelial cell removal, and abolished in the presence of ODQ. SIN-1 caused relaxations of 81±4% (n=6), and ODQ shifted the curve to the right. In vivo, RVP and (RV/LV+S) were increased in hypoxic rats. In all three treated hypoxic groups, pulmonary pressure was reduced, and molsidomine and the combination treatment attenuated (RV/LV+S). Systemic blood pressure was not affected by any treatment or hypoxia. In conclusion, sildenafil and molsidomine relax pulmonary arteries by GC-dependent mechanisms and attenuate the development of PH in chronic hypoxic rats, but there is no synergistic effect of the two drugs.

Department of Pharmacology, Aarhus University, Denmark

P24-08

COMPARATIVE ASPECTS OF IN VITRO REACTIVITY OF RAT AND HUMAN RESPIRATORY SMOOTH MUSCLE.

Tanasie G., Siska IR., Bunu C., Noveanu L., Mihalas G.

Human bronchial fragments were removed from 6 patients undergoing resection for pulmonary carcinoma. We compared the reactivity of preparations, with and without epithelium (denudation was performed with CHAPS, 10mg/ml for 10 minutes). Tracheal preparations were obtained from 12 Sprague Dowley rats, 6 controls and 6 ovalbumin-sensitized animals in order to induce asthmatic-like hypereactivity. Preparations were introduced in isolated organ bath filled Krebs-Henseleit solution, kept at 37°C, bubbled with a 95%O2 and 5%CO2. Contractions and relaxations were recorded usinf a force transducer (FORT 10, WPI Inc.), connected to a computerized data aquisition unit (BIOPAC, AQKNOWLEDGE software).

For human preparations, acetylcholine-induced contractions ranged between 0.185±0.04g at 10-5M, and 0.75±0.03g at 10-3M. Similar effects were registered for metacholine, but carbachol produced a more pronounced effect (p<0.01). In denuded preparations, maximal contraction to acetylcholine was significantly increased (p<0.05).

In healthy rats, the amplitude of acetylcholine-induced contractions was 0.22±0.05g for a 10-7M concentration, and 1.1±0.02g for 10-3M. Metacholine induced similar effects, while carbachol presented a marked contractile (p<0.05). In ovalbumin-sensitized animals, statistically significant differences were noticed between acetylcholine-, metacholine, and carbachol-induced responses.

These results suggest that cholinergic response is similar in the studied species, with differences regarding the amplitude of the contractile response, probably species-related. Epithelium removal could be considered as a model of bronchial hyperreactivity, comparable with sensitization performed in animal models.

University of Medicine and Pharmacy "Victor Babes", Timisoara, Romania

P24-09

AIRWAY RESPONSIVITY IN OVALBUMIN SENSITIZED RATS

Bunu C., Tanasie G., Noveanu L., Siska I., Oancea C., Mihalas G.

The objectives of the our study were:

1) to standardize a sensitization method to ovalbumin

2) to study the contractile response of normal and sensitized rat tracheal smooth muscle, before and after administration of glucocorticoids or synthetic antileukotrienes.

The experiments were performed on 4 groups of Sprague-Dowley adult rats: group I: control; group II: ovalbumin-sensitized rats; group III: ovalbumin-sensitized rats treated with Montelukast, a synthetic leukotriene receptor antagonist; group IV: ovalbumin-sensitized rats treated with glucocorticoids. Tracheal spirals were introduced in organ chambers filled with Krebs-Henseleit solution, bubbled with 95% O2 and 5% CO2, at 37°C. Isometric force was measured using a force transducer and the results were recorded using a computerized data acquisition unit.

In both controls and sensitized rats, muscarinic agonists (10-7 - 10-4M) induced contractions, the relative magnitude of response being: carbachol>metacholine>acetylcholine. In OVA-sensitized rats, reduced contractile reactions were noticed, but the basal tone was significantly higher. In all animals, Pirenzepin administration induced a prominent relaxation of rat precontracted TSM, but its action was more pronounced in sensitized rats (100%). In both controls and sensitized animals, ipratropium bromide induced an augmented relaxation compared to the one induced by Pirenzepin, and shifted the dose-response curve to acetylcholine to the right. In sensitized animals treated with either Montelukast or glucocorticoids, the relaxing response to pirenzepin was significantly higher.

Our research revealed the beneficial effect of synthetic antileukotrienes and glucocorticoids, which restored the normal basal tone and the contractile reserve of TSM, improving the effects of associated bronchodilator agents.

University of Medicine and Pharmacy "Victor Babes", Timisoara, Romania



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