Дисертації з теми "Calcium channel drugs"

Le sombre pronostic du cancer de l’ovaire s’explique notamment par une fréquence importante de résistance à la chimiothérapie conventionnelle présentée par les patientes. La mise en place de stratégies thérapeutiques alternatives à la chimiothérapie, mais aussi la découverte de biomarqueurs prédictifs de la réponse à ce traitement, représentent donc un enjeu majeur pour améliorer la prise en charge de cette pathologie. La chimiorésistance des cellules cancéreuses ovariennes s’explique principalement par leur résistance à l’apoptose, résultant d’un déséquilibre entre les membres pro- et anti-apoptotiques de la famille Bcl-2 qui contrôlent cette mort cellulaire. De ce fait, toutes stratégies capables de moduler le ratio [pro ]/[anti apoptotiques] en faveur des [pro-] restaure efficacement l’apoptose de ces cellules. Or, la signalisation calcique est connue pour réguler l’expression de ces protéines et apparait ainsi comme une cible pertinente pour restaurer l’apoptose des cellules cancéreuses ovariennes chimiorésistantes. Dans ce contexte, nous avons mis en évidence que trois modulateurs du signal calcique sont capables d’induire la mort de ces dernières en association à l’ABT-737, un BH3-mimétique ciblant l’activité de l’anti apoptotique Bcl-xL. Cette sensibilisation à l’ABT-737 est permise par le fait que le carboxyamidotriazole réprime l’expression de l’anti apoptotique Mcl-1 via l’inhibition des courants SOCE, le naftopidil augmente l’expression des protéines pro apoptotiques via l’induction d’un stress du RE ou l’activation de JNK et que la thapsigargine semble préparer à la mort cellulaire grâce à une augmentation de la concentration calcique intracellulaire via STIM1 et, peut-être, via l’induction de l’expression de Noxa. En outre, les acteurs de la signalisation calcique, connus pour subir un remodelage au cours des processus de cancérogenèse pourraient se révéler comme des outils de prédiction de réponse à la chimiothérapie. Dans ce contexte, nous avons mis en évidence que l’expression de la pompe calcique SERCA2 semble jouer le rôle de biomarqueur prédictif de la réponse à la chimiothérapie des patientes atteintes d’un cancer de l’ovaire<br>The poor prognosis of ovarian cancer is mainly explained by a high rate of resistance to conventional chemotherapy presented by patients. Therefore, discovery of both alternative therapeutic strategies to chemotherapy and predictive biomarkers for response to this treatment represent a major challenge for improving the management of this pathology. Chemoresistance of ovarian cancer cells is mainly due to their resistance to apoptosis, resulting from an imbalance between the pro- and anti-apoptotic members of the Bcl-2 family that control this type of cell death. Thus, all strategies able to modulate the [pro]/[anti-apoptotic] protein ratio in favor of [pro-] effectively restore apoptosis in these cells. However, calcium signaling is known to regulate the expression of these proteins and thus appears to be a relevant target for restoring apoptosis in chemoresistant ovarian cancer cells. In this context, we have shown that three calcium signal modulators are able to induce the death of these cells in association with ABT-737, a BH3-mimetic targeting the activity of the anti-apoptotic Bcl-xL. This sensitization to ABT-737 is enabled by the fact that carboxyamidotriazole represses the expression of the anti apoptotic Mcl-1 via the inhibition of SOCE currents, naftopidil increases pro-apoptotic protein expression via ER stress induction or JNK activation and thapsigargin seems to prepare cell death through increasing intracellular calcium concentration via STIM1 and, maybe, through Noxa expression induction. In addition, players of the calcium signaling toolkit, known to undergo remodeling during carcinogenesis could be proven as tools for predicting response to chemotherapy. In this context, we have shown that the expression of the calcium pump SERCA2 seems to play a role as a predictive biomarker for response to chemotherapy of patients with ovarian cancer

The current recommendations by the American Heart Association for health promotion are that all persons should partake in regular physical activity in order to reduce the risk of cardiovascular disease. Regular physical exercise reduces blood pressure and is an important component of the management of hypertension. It is therefore important that patients with hypertension participate in habitual physical exercise. Many hypertensive patients who exercise will require anti-hypertensive medication. However, some antihypertensive agents cause fatigue during exercise. In order for patients to gain the full benefits of an active lifestyle, it is important that the prescribed antihypertensive agent does not prevent them performing and enjoying sustained exercise. It has been well documented that β-blockers cause premature fatigue during physical exercise. The effects on exercise performance of other first line antihypertensive medications, such as calcium channel antagonists have not been extensively investigated. In particular, the effects of these agents on prolonged submaximal exercise endurance have not been well studied. The object of this thesis was to compare the effects of isradipine, a dihydropyridine calcium channel antagonist, to those of atenolol, a β₁-selective antagonist, on maximal and submaximal exercise performance and on short duration high-intensity exercise in physically active hypertensive patients. The study design was a crossover trial where drug treatments were double blinded and randomised. Physically active volunteers with mild to moderate hypertension were recruited. 11 subjects performed i) progressive exercise to exhaustion for determination of maximal oxygen consumption (VO₂max), maximal work load and cardiorespiratory responses to maximal exercise, ii) prolonged submaximal exercise for determination of exercise endurance, cardiorespiratory responses and ratings of perceived exertion (APE), and iii) short duration, high intensity exercise consisting of a 30 second maximal exercise test (Wingate test) to determine skeletal muscle power output, following 4 weeks ingestion of isradipine (2.5mg bd), atenolol (50mg bd) or placebo. Diastolic blood pressure at rest was reduced by both atenolol and isradipine, but was lowered to a greater extent by atenolol (83.3 vs 89.0 vs 96.1 mmHg, atenolol vs isradipine vs placebo, p<.0005). Systolic blood pressure at rest tended to be similarly reduced by both agents, but was significantly reduced during maximal and submaximal exercise by atenolol only (p<.001, atenolol vs isradipine, placebo). Heart rate at rest and during maximal and submaximal exercise was decreased by atenolol only (p<.0005, atenolol vs isradipine, placebo). Maximal exercise performance was reduced after atenolol ingestion compared to placebo but not after isradipine ingestion. Peak workload achieved during the maximal exercise test was decreased after atenolol but unchanged after isradipine ingestion (214 vs 243 W, atenolol vs placebo, p<.01). Similarly, VO₂max was reduced after atenolol compared to placebo but was unchanged after isradipine ingestion (33.6 vs 36.4, 33.6 vs 36.1 mlO₂/kg/min, atenolol vs placebo, atenolol vs isradipine, p<.05). Both atenolol and isradipine ingestion reduced submaximal endurance time compared to placebo (27.8 vs 46.4, 34.4 vs 46.4 min, atenolol vs placebo, isradipine vs placebo, p<.005), and increased rating of perceived exertion (APE) after 30 min of submaximal exercise (p<.05). Submaximal oxygen consumption (VO₂), ventilation, respiratory exchange ratio (REA) and blood lactate, glucose and free fatty acid concentrations were not altered after the ingestion of either agent. Neither agent influenced peak skeletal muscle power, total work done, or rate of fatigue during the Wingate test compared to placebo. The results of these studies indicate that impaired performance and increased RPE during submaximal exercise after ingestion of either atenolol or isradipine is not due to alterations of ventilation, VO₂, RER, or blood lactate, glucose and free fatty acid concentrations during prolonged submaximal exercise. Similarly, reduced submaximal exercise performance after atenolol or isradipine ingestion is not due to factors which would also limit the ability of skeletal muscle to perform short duration, high intensity exercise before a bout of prolonged exercise. This study demonstrates that prolonged submaximal exercise testing can reveal an impairment in exercise performance after ingestion of antihypertensive medication which is not evident during maximal exercise testing. This finding is important as prolonged submaximal exercise is the form of exercise which most hypertensive patients actually perform. Further research is required on the effects of anti-hypertensive medications on submaximal exercise performance before firm recommendations can be made regarding medications most suitable for the physically active hypertensive patient. The results of these and other studies indicate that it is not yet possible to make claims that the calcium channel antagonist agents are without effect on physical exercise performance in physically active hypertensive patients.

BK channels are well studied targets of acute ethanol action. They play a prominent role in neuronal excitability and have been shown to play a significant role in behavioral ethanol tolerance in invertebrates. The focus of my work centers on the effects of alcohol on the BK channel and comprises studies that examine how subcellular location affects acute ethanol sensitivity and how duration of acute alcohol exposure impacts the development of rapid tolerance. My results also provide potential mechanisms which underlie acute sensitivity and rapid tolerance. I first explore BK channel sensitivity to ethanol in the three compartments (dendrite, cell body, and nerve terminal) of magnocellular neurons in the rat hypothalamic-neurohypophysial (HNS) system. The HNS system provides a particularly powerful preparation in which to study the distribution and regional properties of ion channel proteins because the cell bodies are physically separated from the nerve terminals. Using electrophysiological and immunohistochemical techniques I characterize the BK channel in each of the three primary compartments and find that dendritic BK channels, similar to somatic channels, but in contrast to nerve terminal channels, are insensitive to alcohol. Furthermore, the gating kinetics, calcium sensitivity, and iberiotoxin sensitivity of channels in the dendrite are similar to somatic channels but sharply contrast terminal channels. The biophysical and pharmacological properties of somatodendritic vs. nerve terminal channels are consistent with the characteristics of exogenously expressed αβ1 vs. αβ4 channels, respectively. Therefore, one possible explanation for my findings is a selective distribution of β1 subunits to the somatodendritic compartment and β4 subunits to the terminal compartment. This hypothesis is supported immunohistochemically by the appearance of distinct punctate β1 or β4 channel clusters in the membrane of somatodendritic or nerve terminal compartments, respectively. In conclusion, I found that alcohol sensitivity of BK channels within the HNS system is dependent on subcellular location and postulate that β-subunits modulate ethanol sensitivity of HNS BK channels. In the second and primary focus of my thesis I explore tolerance development in the striatum, a brain region heavily implicated in addiction. Numerous studies have demonstrated that duration of drug exposure influences tolerance development and drug dependence. To further elucidate the mechanisms underlying behavioral tolerance I examined if BK channel tolerance was dependent on duration of alcohol exposure using patch clamp techniques in cultured striatal neurons from P8 rats. I found that persistence of rapid tolerance is indeed a function of exposure time and find it lasts surprisingly long. For example, after a 6 hr exposure to 20 mM ethanol, acute sensitivity was still suppressed at 24 hrs withdrawal. However, after a 1 or 3 hr exposure period, sensitivity had returned after only 4 hrs. I also found that during withdrawal from a 6 hr but not a 3 hr exposure the biophysical properties of BK channels change and that this change is correlated with an increase in mRNA levels of the alcohol insensitive STREX splice variant. Furthermore, BK channel properties during withdrawal from a 6 hr exposure to alcohol closely parallel the properties of STREX channels exogenously expressed in HEK293 cells. In conclusion I have established that BK channels develop rapid tolerance in striatal neurons, that rapid tolerance is dependent upon exposure protocol, and is surprisingly persistent. These findings present another mechanism underlying BK channel tolerance and possibly behavioral tolerance. Since these phenomena are dependent on duration of drug exposure my results may find relevance in explaining how drinking patterns impact the development of alcohol dependence in humans.

Alcoholism is responsible for more than 6% of deaths internationally per annum. The development of acute tolerance to ethanol (EtOH) is a critical component of alcoholism. Previous studies identified large conductance calcium-activated potassium (BK) channels as potential EtOH targets in a variety of species and cells. In order to elucidate mechanisms underlying tolerance development, I used inside-out patch clamp techniques to measure EtOH induced changes in channel activity (measured as open probability) of hSlo, hSlo+β1, and hSlo+β4 channels exogenously expressed in HEK 293 cells. I show that the human BK channels have subunit dependent responses to acute application of EtOH, and the magnitude of potentiation was dependent on the concentration of ethanol used and the type of β-subunit expressed. In addition the subunit dependent effects on the channels were a function of cytosolic calcium concentration. Furthermore, to determine if BK channels in ripped-off patches can become tolerant to EtOH, I monitored changes in channel activity in response to a second application of the drug, 10-minutes after washing-out the first exposure. I found that channels were less responsive to the second exposure, indicative of tolerance. I examined long-term consequences of EtOH exposure by repeating these experiments on cells cultured in 25 mM EtOH in the culture medium for 24-hours. Under these conditions, all three channel types show chronic tolerance has developed as revealed by the response to acute EtOH applications. Subunit-dependent differences to the development of acute tolerance were apparent, however. In response to a second application to EtOH, hSlo+β4 channels were now inhibited. Overall, these results indicate that BK channels respond to and develop tolerance to EtOH in the absence of cellular context, suggesting the possibility that alcohol tolerance within organisms may be in part mediated by changes imparted by EtOH on BK channels directly.

The rapid discovery of novel therapeutic agents, targeting the specific mechanism of cancer progression, invasion and angiogenesis, necessitates the development and validation of efficient techniques to assess the therapeutic efficacy of these drugs in vivo. Recently the development of dedicated PET scanners for the imaging of small animals, such as the microPET system (CTI Concorde R4), has allowed for the high-resolution functional and molecular imaging of murine and rodent models of disease. This study, investigates the ability of microPET imaging, using the 18F labelled 2-fluoro-2-deoxyglucose (FDG) PET tracer, to detect the therapeutic efficacy of novel targeted therapies in a rat model of glioma. This technique potentially allows for the rapid and high-throughput assessment of tumour response and evaluation of efficacy of such therapeutic agents in vivo at the pre-clinical stage and will, consequently, facilitate the translation of these novel drugs from the discovery to the clinical phases.<br>La découverte accélérée de nouvelles molécules thérapeutiques qui ciblent lesmécanismes de progression du cancer tels que l'invasion et l'angiogenèse, nécessite lamise au point et la validation de techniques efficaces qui permettent d'évaluer l'efficacitéthérapeutique de ces agents in vivo. Le développement récent des scanners detomographie à émission de positron (TEP) dédiés à l'imagerie de petits animaux(microPET, CT! Concorde R4), permet aujourd'hui d'obtenir une image fonctionnelle etmoléculaire de haute résolution des modèles rongeurs. Cette étude s'intéresse au potentieldu 18F-2-fluoro-2-deoxyglucose (FDG) en utilisant l'imagerie microPET dansl'évaluation de l'efficacité de nouveaux agents thérapeutiques dans un modèle de gliomechez le. rat. Cette technique pourrait éventuellement mener à une évaluation rapide et àgrande échelle de la réponse tumorale, ainsi que la mesure de l'efficacité d'agentsthérapeutiques in vivo au stade d'étude préclinique. Globalement, cette étude a pour butde faciliter la transition entre la découverte de nouvelles molécules thérapeutiques et leursapplications cliniques.

博士<br>臺北醫學大學<br>藥學研究所<br>95<br>Photosensitivity is a commonly adverse effect of drugs. The purpose of the first part of this study is focus on the photodegradation of nicardipine. When nicardipine was exposed to the Hg lamp, eight photoproducts of nicardipine were identification by LC/MS. The main degraded product was a pyridine analogue (NIC-7). Nicardipine apparently undergoes a series of nitro group photo-reduction pathways under irradiation leading to a complex formation of mainly the reduced products. A reaction scheme of nicardipine was proposed. The second part, gives a study on the photochemical behavior when NSAIDs (flurbiprofen and indomethacin) in alcoholic solvents are exposed to Hg lamps. GC/MS and LC/MS were applied to determine the structure of photoproducts. In addition, some pharmacological effects were also examined. In total, ten and four photoproducts derived from flurbiprofen and indomethacin methanolic samples, respectively, were identified by GC/MS and LC/MS. Furthermore, the reaction schemes of flurbiprofen and indomethacin in methanol are proposed. As to the study of pharmacological effects, results suggested that among all the related photoproducts, Indomethacin stand out and showed the strongest hydroxyl radical-scavenging effect with an IC50 of 65 µM and the strongest xanthine oxidase inhibitory effect with an IC50 of 86 µM. We also found that the methyl ester derivatives of indomethacin (IN-3) could more-potently inhibit PGE2 and NO production and iNOS and COX 2 protein expression from LPS-stimulated RAW 264.7 cells than indomethacin, similar to the effect of a typical NSAID. The cytotoxic effects of the test samples were measured using the MTT assay. The results showed that IN-3 with an IC50 value maintained at 36.9 ?慊/mL for 12 h that exhibited stronger cytotoxicity than indomethacin in HL-60 cells. Moreover, IN-3 caused apoptotic bodies, DNA fragmentation, and enhanced PARP and pro-caspase 3 degradation in HL-60 cells as determined by a series of biochemical analyses. The above results indicated that the photoproduct, IN-3, had stronger anti-inflammatory in LPS-stimulated RAW 264.7 cells and cytotoxicity effects in HL-60 cells than the parent drug, indomethacin.

The aim of the study was to examine the use of a repolarizing calcium dependent potassium current K(Ca) in neurons to describe the actions of a group of novel compounds with potential Class ifi actions on cardiac cells. This was accomplished by determination of the correlation between potency of the agents to block single channel K(Ca) and potency to prolong effective refractory period (ERP) in heart. If a positive correlation could be established then elucidation of mechanisms of drug actions on single channel K(Ca) could have utility in the description of drug actions on repolarizing K+ currents in heart. At present the low unitary conductance of transient outward and delayed rectifier K+ channels precludes a mechanistic analysis of drug actions on cardiac cells. Initial experiments included the measurements of the single channel properties of the K(Ca) using inside-out patches obtained from cultured hippocampal neurons. The channel conductances, with physiological (SK+ and 140K+ across patches) and symmetrical (140 K+ across patches) were 110 pS and 170 pS respectively. A requirement of 4 µM internal calcium was necessary to maintain maximal channel activity with a threshold for K(Ca) activation at 0.7 µM. At low internal calcium concentration, depolarization increased the probability of channel openings. The effect was found to be solely dependent on the voltage-sensitive increase of the channel opening frequency; mean open times of the channel were not dependent on patch potential. The unitary K(Ca) is the microscopic basis for the macroscopic repolarizing current Ic in hippocampal neurons. Ic is responsible for the late repolarization phase and the early afterhyperpolarization (AHP) phase associated with the neuronal action potential. It was of interest to first determine the effects on Ic of an agent, tedisamil, with known Class ifi activity in heart. The results showed the drug both prolonged the neuronal action potential and eliminated the subsequent AHP phase. The primary set of experiments involved the investigations of the effects of 18 RSD novel compounds on unitary K(Ca). All the compounds, except for three, were effective in exhibiting rapid transitions in the K(Ca) from the opening state to a non-conducting state in the inside-out patches. The mean open times of open events were reduced but the closed time durations and the channel amplitudes were not changed. The potency of the effect of the RSD compounds on the K(Ca) was determined as the concentration required to halve the mean open time relative to control value. According to this index the compounds were categorized into five different groups based on potency to decrease mean open time of K(Ca). In addition the actions of five of the RSD compounds on K(Ca) were examined using outside-out patches excised from neurons. The potency of the RSD compounds on the neuronal K(Ca) was compared with their potency in inhibiting repolarizing K+ currents in the rat whole heart. The index for potency used in the whole heart experiments was the concentrations of the compounds required to increase the effective refractory period by 25%. Of the 18 compounds tested, 3 were found to be inactive (no obvious effect for concentration below 5OµM) on properties ofK(Ca). These same 3 agents also were ineffective in whole heart (in excess of 2OpM). For 13 of the remaining 15 agents, a positive correlation was found (with a correlation coefficient r of 0.71) between potency to block K(Ca) and potency to prolong ERP in whole heart. However, with 2 agents there was no apparent correlation for actions in the neurons and the heart. It was also established that drugs with persistent effects on K(Ca) (likely due to prolonged bonding to membrane sites) were also long-lasting in whole heart experiment.

Invertebrate T-type calcium channels cloned from the great pond snail, Lymnaea Stagnalis (LCav3) possess highly sodium permeant ion channel currents by means of alternative splicing of exon 12. Exon 12 is located on the extracellular turret and the descending helix between segments 5 and segments 6, upstream of the ion selectivity filter in Domain II. Highly-sodium permeant T-type channels are generated without altering the selectivity filter locus, the primary regulatory domain known to govern ion selectivity for calcium and sodium channels. Comparisons of exon 12 sequences between invertebrates and vertebrate T-type channels reveals a conserved pattern of cysteine residues. Calcium-selective mammalian T-type channels possess a single cysteine in exon 12 in comparison to invertebrate T-type channels with either a tri- or penta- cysteine framework. Cysteine residues in exon 12 were substituted with a neutral amino acid, alanine in LCav3 channels harbouring exon 12a and 12b to mimic the turret structure of vertebrate T-type channels. The results generated T-type channels that were even more sodium-permeable than the native T-type channels in snails. Furthermore, permeant divalent ions similar in structure to calcium (eg. barium) were unable to sufficiently block the monovalent ion current of channels lacking cysteines in Domain II, suggesting that the pore is highly sodium permeant, and has weak affinity and block by permeant divalent ions other than calcium. Besides ion selectivity, the cysteine mutated T-type channels were 10 to 100 fold more sensitive to inhibition by nickel and zinc, respectively. The cysteine mutation data highly suggests that the cysteines form an extracellular structure that regulates ion selectivity and shields T-type channels from block by nickel and zinc. In addition, we replaced exon 12 from the sodium permeant snail T-type channel with exon 12 from human Cav3.2 channels. The snail T-type channel with exon 12 from human T-type channels produced a T-type channel that was modestly sodium permeable, but did not confer the high calcium permeability of Cav3.2 channels. These findings suggest that the cysteine containing extracellular domains in exon 12 are not sufficient to generate calcium selective channels similar to human Cav3.2 and likely work in concert with other extracellular domains to regulate the calcium or sodium selectivity of T-type channels.

The present research is focused on the topic of inflammation-drug interaction. Inflammation complicates many human diseases and conditions ranging from obesity to cancer. Therefore, the study of the effect of inflammation on drug pharmacokinetics and pharmacodynamics is pivotal. First, we tested the hypothesis that controlling inflammation using valsartan can restore the previously reported altered verapamil pharmacokinetics and pharmacodynamics. Such an effect is expected due to the anti-inflammatory properties of angiotensin II inhibition. Inflammation resulted in L-type calcium channel target protein (Cav1.2) downregulation and reduced verapamil potency in pre-adjuvant arthritis rat model. Valsartan treatment reversed the observed downregulation of L-type calcium channels thereby enhancing verapamil potency. This beneficial interaction, once proven in humans, may be of value in cardiac patients with superimposing inflammatory diseases. Second, we investigated whether the response to verapamil is reduced in experimentally induced acute myocardial injury (AMI) in rats. AMI caused a 75% reduction in verapamil potency and Cav1.2 target protein downregulation. If extrapolated to humans, our observations may suggest that L-type calcium channel downregulation can contribute, at least in part, to the poor outcome in myocardial infarction patients treated with calcium channel blockers (CCBs). Third, we studied the effect of obesity on the pharmacological response of CCBs in children with renal disease. Our data indicated that obese children are less responsive to CCBs than non-obese ones. Therefore, obesity should be considered when initiating antihypertensive drug therapy in children. Last, we were interested in finding out if the expression of other target genes is also altered by inflammation. We used real time polymerase chain reaction, after determination of the best housekeeping gene to be used as an internal control. Inflammation resulted in significant alterations of several molecular targets and transporters affecting the pharmacokinetics and pharmacodynamics of drugs. These findings may provide an insight into the effect of inflammation on drug targets and modulators of disease pathogenesis. In conclusion, inflammation is a missed ring in the chain of therapy. The research presented in this thesis will add to the inflammation-drug interaction field important findings that will help understanding the role of inflammation in pharmacotherapy outcomes.<br>pharmaceutical sciences

by Ai-Dong Qi.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 1997.<br>Includes bibliographical references (p. 219-256).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Mode of access: World Wide Web.

碩士<br>高雄醫學大學<br>藥學系碩士在職專班<br>103<br>Background: Previous case reports revealed that concomitant therapy with calcium channel blockers and macrolides resulted in hypotension. In 2012, the U.S. FDA issued a warning to remind physicians that the combination of clarithromycin and calcium channel blockers may cause severe hypotension. Drug-drug interactions not only affect the effectiveness, but also cause adverse effects, especially in cardiovascular drugs. Because a lot of cardiovascular drugs are metabolized by cytochrome P450 enzyme systems, simultaneous use with CYP 3A4 inhibitors or inducers, will lead to fluctuations of therapeutic levels, and further resulted in some adverse effects. Contrary to azithromycin, erythromycin and clarithromycin have inhibitory activity of cytochrome P450 3A4 (CYP 3A4). Therefore, co-administration with some calcium channel blockers which are the substrates of CYP 3A4 system will increase the risk of hypotension. Consequently, while hypotension occurs, poor kidney perfusion may also be a concern. Objective: We conducted a population-based cohort study to investigate the incidence of acute kidney injury, hypotension and shock from the possible drug-drug interaction of calcium channel antagonists-macrolides. Methods: The study used the 2005 National Health Insurance Research Database (NHIRD) from 2000 to 2012. We identified patients who had concurrent usage of calcium channel blockers and macrolides in 2002~2012. According to CYP 3A4 inhibitor activity, users of erythromycin/clarithromycin were in the treatment group, and azithromycin users were the control group. The incidences of hypotension, shock, and acute kidney injury after concurrent usage were identified. The propensity scores (PS) weighting were adapted in the statistical analysis. Results: In the period between 2000~2012, those combinations at the same prescription,were frequently prescribed by internists, accounting for 59.37%% of all, and it had more frequency of occurrence in local community hospitals (60.53%) than in the clinic. We also identified 1,774 patients who received a coprescription with calcium channel blockers and macrolides in the period between 2002~2012, including 1,407 patients in erythromycin/clarithromycin group and 367 patients in azithromycin group. The incidence of acute kidney injury in azithromycin group (7.08%) was higher than in erythromycin/ clarithromycin group (3.20%) with odds ratio (OR) of 0.43 (95% CI: 0.26~0.71). But the incidence of hypotension or shock was not statistical significance from the two groups (OR: 0.55, 95% CI: 0.29~1.05). However, in azithromycin group, there were more comorbidities, and more renal disease patients. Therefore, propensity score was used to balance the two groups. In those who had underlying disease with renal disease, the incidence of acute kidney injury outcome in erythromycin/ clarithromycin group was 14.52%, and in azithromycin group was 12.70% (p= 0.73, weighted OR: 1.77, 95% CI: 0.98~3.18). Similarly, incidences of hypotension or shock were respectively 3.23%, 6.35% (p= 0.45, weighted OR: 1.50, 95% CI: 0.61~3.69). Furthermore, in our study, older age, multiple comorbidities, chronic renal disease, and the longer length of combinated days seemed to relate between acute kidney injury in erythromycin/clarithromycin group. Conclusions: The finding did not support the theory that combination with azithromycin would be more risk than erythromycin/clarithromycin group. There was no statistical significance in incidences of hypotension or shock between two groups in 18 years older Taiwanese.

The objective of these experiments was to investigate the actions of a number of novel putative class III antiarrhythmic agents on the large conductance calcium-activated potassium channel (BK ) in cultured hippocampal neurons. The experiments were carried out in three steps. In the first set of experiments (BK)ca channel, isolated from cultured rat hippocampal CA1 neuronal somatic membrane, was first identified and its physiological and pharmacological properties were characterized at a single channel level. This (BKca ) channel had an average single channel conductance of 80 pS with physiological transmembrane K+ (140 mM inside and 5 mM outside). It was very selective to K+over Na+, a ratio of 100 to 7 was determined from this experiment. Calcium, at the internal side of the membrane, was necessary to activate the BKca channel. With low internal calcium concentration, depolarization could promote the rate of channel openings. An e-fold increase in p0 was found with 1 µ M internal calcium and 15 mV depolarization. With 200 µM internal calcium, p0 was virtually independent of voltage. 0.1 mM external TEA showed fast blockade of this channel. Internal TEA and 4-AP (internal or external) showed no effect on BKca. In the second set of experiments, the actions of putative class III drugs on BKca was studied. The drugs were RP-62719, UK-68798, tedisamil (KC-8857) and risotilide (WY-48986) at concentrations 0.1 ~ 10 µ M. All these agents, applied both to the inside or to the outside of the patch membrane, resulted in the opening of the BKca channel to exhibit rapid flicking from open to nonconducting levels. This effect was dose-dependent and for KC-8857 and UK-68798 was evident at concentrations of 0.1 µ M. The blocking rate constants were determined from a simple open channel blockade scheme and were not dependent on voltages. Single channel conductance and ionic selectivity were not affected by the drugs. The potencies for channel block of the drugs acting either externally or internally were in the order UK-68798>tedisamil>RP-62719> risotilide with UK-68798 reducing the mean open time of BKca by one-half at a concentration near 0.4 µ M. In the final set of experiments, the thermodynamics associated with RP- 62719 block BKca channel was studied in order to better understand the molecular mechanisms of channel block. The Q10 associated with the channel mean open time was found to be 2.2 with 5 µ M RP-62719 at the inner surface of the patch membrane. The blocking and unblocking rate constants were determined using the simple open channel block scheme. Thermodynamic analysis, using transition rate theory, showed that the blocking rate constant was associated with a large increase in entropy. The relatively high temperature dependence for channel blockade was not consistent with a rate-limiting process established by simple diffusion of the agent to a channel blocking site. Channel block may involve conformational changes in the channel protein as a consequence of hydrophobic interactions between drug and channel sites.

Kar Wing To.<br>Thesis submitted in: December 1999.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2000.<br>Includes bibliographical references (leaves 200-223).<br>Abstracts in English and Chinese.<br>Acknowledgements --- p.i<br>List of Abbreviations --- p.ii<br>Abstract --- p.v<br>撮要 --- p.viii<br>List of Tables --- p.x<br>List of Figures --- p.xi<br>Contents --- p.xv<br>Chapter CHAPTER 1 --- INTRODUCTION<br>Chapter 1.1 --- The General Characteristics of Glial Cells --- p.1<br>Chapter 1.1.1 --- Astrocytes --- p.1<br>Chapter 1.1.2 --- Oligodendrocytes --- p.5<br>Chapter 1.1.3 --- Microglial --- p.6<br>Chapter 1.2 --- Brain Injury and Astrocyte Proliferation --- p.6<br>Chapter 1.3 --- Reactive Astrogliosis and Glial Scar Formation --- p.9<br>Chapter 1.4 --- Astrocytes and Immune Response --- p.10<br>Chapter 1.5 --- Cytokines --- p.10<br>Chapter 1.5.1 --- Cytokines and the Central Nervous System (CNS) --- p.12<br>Chapter 1.5.2 --- Cytokines and brain injury --- p.13<br>Chapter 1.5.3 --- Cytokines-activated astrocytes in brain injury --- p.13<br>Chapter 1.5.4 --- Tumour Necrosis Factor-a --- p.14<br>Chapter 1.5.4.1 --- Types of TNF-α receptor and their sturctures --- p.16<br>Chapter 1.5.4.2 --- Binding to TNF-α --- p.17<br>Chapter 1.5.4.3 --- Different Roles of the TNF-a Receptor Subtypes --- p.17<br>Chapter 1.5.4.4 --- Role of TNF-α and Brain Injury --- p.19<br>Chapter 1.5.4.5 --- TNF-α Stimulates Proliferation of Astrocytes and C6 Glioma Cells --- p.23<br>Chapter 1.5.5 --- Interleukin-1 (IL-1) --- p.26<br>Chapter 1.5.5.1 --- Interleukin-1 and Brain Injury --- p.27<br>Chapter 1.5.6 --- Interleukin-6 (IL-6) --- p.28<br>Chapter 1.5.6.1 --- Interleukin-6 and brain injury --- p.29<br>Chapter 1.5.7 --- γ-Interferon (γ-IFN) --- p.30<br>Chapter 1.5.7.1 --- γ-Interferon and Brain Injury --- p.30<br>Chapter 1.6 --- Ion Channels and Astrocytes --- p.31<br>Chapter 1.6.1 --- Roles of Sodium Channels in Astrocytes --- p.33<br>Chapter 1.6.2 --- Role of Potassium Channels in Astrocytes --- p.33<br>Chapter 1.6.3 --- Importance of Calcium Ion Channels in Astrocytes --- p.34<br>Chapter 1.6.3.1 --- Function of Cellular and Nuclear Calcium --- p.34<br>Chapter 1.6.3.2 --- Nuclear Calcium in Cell Proliferation --- p.36<br>Chapter 1.6.3.3 --- Nuclear Calcium in Gene Transcription --- p.36<br>Chapter 1.6.3.4 --- Nuclear Calcium in Apoptosis --- p.38<br>Chapter 1.6.3.5 --- Spatial and Temporal Changes of Calcium-Calcium Oscillation --- p.39<br>Chapter 1.6.3.6 --- Calcium Signalling in Glial Cells --- p.39<br>Chapter 1.6.3.7 --- Calcium Channels in Astrocytes --- p.41<br>Chapter 1.6.3.8 --- Relationship Between [Ca2+]i and Brain Injury --- p.43<br>Chapter 1.6.3.9 --- TNF-α and Astrocyte [Ca2+]i --- p.45<br>Chapter 1.6.3.10 --- Calcium-Sensing Receptor (CaSR) --- p.46<br>Chapter 1.7 --- Protein Kinase C (PKC) Pathways --- p.49<br>Chapter 1.7.1 --- PKC and Brain Injury --- p.50<br>Chapter 1.7.2 --- Role of Protein Kinase C Activity in TNF-α Gene Expression in Astrocytes --- p.51<br>Chapter 1.7.3 --- PKC and Calcium in Astrocytes --- p.52<br>Chapter 1.8 --- Intermediate Early Gene (IEGs) --- p.54<br>Chapter 1.8.1 --- IEGs Expression and Brain Injury --- p.54<br>Chapter 1.8.2 --- IEGs Expression and Calcium --- p.55<br>Chapter 1.9 --- The Rat C6 Clioma Cells --- p.56<br>Chapter 1.10 --- The Aim of This Project --- p.58<br>Chapter CHAPTER 2 --- MATERIALS AND METHODS<br>Chapter 2.1 --- Materials --- p.61<br>Chapter 2.1.1 --- Sources of the Chemicals --- p.61<br>Chapter 2.1.2 --- Materials Preparation --- p.65<br>Chapter 2.1.2.1 --- Rat C6 Glioma Cell Line --- p.65<br>Chapter 2.1.2.2 --- C6 Glioma Cell Culture --- p.65<br>Chapter 2.1.2.2.1 --- Complete Dulbecco's Modified Eagle Medium (CDMEM) --- p.65<br>Chapter 2.1.2.2.2 --- Serum-free Dulbecco's Modified Eagle Medium --- p.66<br>Chapter 2.1.2.3 --- Phosphate Buffered Saline (PBS) --- p.66<br>Chapter 2.1.2.4 --- Recombinant Cytokines --- p.67<br>Chapter 2.1.2.5 --- Antibodies --- p.67<br>Chapter 2.1.2.5.1 --- Anti-TNF-Receptor 1 (TNF-R1) Antibody --- p.67<br>Chapter 2.1.2.5.2 --- Anti-TNF-Receptor 2 (TNF-R2) Antibody --- p.67<br>Chapter 2.1.2.6 --- Chemicals for Signal Transduction Study --- p.68<br>Chapter 2.1.2.6.1 --- Calcium Ionophore and Calcium Channel Blocker --- p.68<br>Chapter 2.1.2.6.2 --- Calcium-Inducing Agents --- p.68<br>Chapter 2.1.2.6.3 --- Modulators of Protein Kinase C (PKC) --- p.69<br>Chapter 2.1.2.7 --- Reagents for Cell Proliferation --- p.69<br>Chapter 2.1.2.8 --- Reagents for Calcium Level Measurement --- p.70<br>Chapter 2.1.2.9 --- Reagents for RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.71<br>Chapter 2.1.2.10 --- Sense and Antisense Used --- p.72<br>Chapter 2.1.2.11 --- Reagents for Electrophoresis --- p.74<br>Chapter 2.2 --- Methods --- p.74<br>Chapter 2.2.1 --- Maintenance of the C6 Cell Line --- p.74<br>Chapter 2.2.2 --- Cell Preparation for Assays --- p.75<br>Chapter 2.2.3 --- Determination of Cell Proliferation --- p.76<br>Chapter 2.2.3.1 --- Determination of Cell Proliferation by [3H]- Thymidine Incorporation --- p.76<br>Chapter 2.2.3.2 --- Measurement of Cell Viability Using Neutral Red Assay --- p.77<br>Chapter 2.2.3.3 --- Measurement of Cell Proliferation by MTT Assay --- p.77<br>Chapter 2.2.3.4 --- Protein Assay --- p.78<br>Chapter 2.2.3.5 --- Data Analysis --- p.79<br>Chapter 2.2.3.5.1 --- The Measurement of Cell Proliferation by [3H]- Thymidine Incorporation --- p.79<br>Chapter 2.2.3.5.2 --- The Measurement of Cell growth in Neutral Red and MTT Assays --- p.79<br>Chapter 2.2.3.5.3 --- The Measurement of Cell Proliferationin Protein Assay --- p.79<br>Chapter 2.2.4 --- Determination of Intracellular Calcium Changes --- p.80<br>Chapter 2.2.4.1 --- Confocal Microscopy --- p.80<br>Chapter 2.2.4.1.1 --- Procedures for Detecting Cell Activity by CLSM --- p.81<br>Chapter 2.2.4.1.2 --- Precautions of CLSM --- p.82<br>Chapter 2.2.5 --- Determination of Gene Expression by Reverse- Transcription Polymerase Chain Reaction (RT-PCR) --- p.83<br>Chapter 2.2.5.1 --- RNA Preparation --- p.83<br>Chapter 2.2.5.1.1 --- RNA Extraction --- p.83<br>Chapter 2.2.5.1.2 --- Measurement of RNA Yield --- p.84<br>Chapter 2.2.5.2 --- Reverse Transcription (RT) --- p.84<br>Chapter 2.2.5.3 --- Polymerase Chain Reaction (PCR) --- p.85<br>Chapter 2.2.5.4 --- Separation of PCR Products by Agarose Gel Electrophoresis --- p.85<br>Chapter 2.2.5.5 --- Quantification of Band Density --- p.86<br>Chapter CHAPTER 3 --- RESULTS<br>Chapter 3.1 --- Effects of Different Drugs on C6 Cell Proliferation --- p.87<br>Chapter 3.1.1 --- Effects of Cytokines on C6 Cell Proliferation --- p.87<br>Chapter 3.1.1.1 --- Effect of TNF-α on C6 Proliferation --- p.88<br>Chapter 3.1.1.2 --- Effects of Other Cytokines on C6 Cell Proliferation --- p.92<br>Chapter 3.1.2 --- The Signalling Pathway of TNF-α induced C6 Cell Proliferation --- p.92<br>Chapter 3.1.2.1 --- The Involvement of Calcium Ions in TNF-α-induced C6Cell Proliferation --- p.95<br>Chapter 3.1.2.2 --- The Involvement of Protein Kinase C in TNF-α- induced C6 Cell Proliferation --- p.96<br>Chapter 3.1.3 --- Effects of Anti-TNF Receptor Subtype Antibodies on C6 Cell Proliferation --- p.102<br>Chapter 3.2 --- The Effect of in Tumour Necrosis Factor-α on Changesin Intracellular Calcium Concentration --- p.102<br>Chapter 3.2.1 --- Release of Intracellular Calcium in TNF-α-Treated C6 Cells --- p.104<br>Chapter 3.2.2 --- Effects of Calcium Ionophore and Calcium Channel Blocker on TNF-α-induced [Ca2+]i Release --- p.107<br>Chapter 3.2.3 --- Effects of Other Cytokines on the Change in [Ca2+]i --- p.109<br>Chapter 3.2.4 --- The Role of PKC in [Ca2+]i release in C6 Glioma Cells --- p.109<br>Chapter 3.2.4.1 --- Effects of PKC Activators and Inhibitors on the Changes in [Ca2+]i --- p.114<br>Chapter 3.3 --- Determination of Gene Expression by RT-PCR --- p.114<br>Chapter 3.3.1 --- Studies on TNF Receptors Gene Expression --- p.117<br>Chapter 3.3.1.1 --- Effect of TNF-α on TNF Receptors Expression --- p.117<br>Chapter 3.3.1.2 --- Effects of Other Cytokines on the TNF Receptors Expression --- p.119<br>Chapter 3.3.1.3 --- Effects of Anti-TNF Receptor Subtype Antibodies on the TNF-a-induced Receptors Expression --- p.121<br>Chapter 3.3.1.4 --- Effect of Calcium Ions on TNF Receptors Expression --- p.121<br>Chapter 3.3.1.4.1 --- Effect of Calcium Ionophore on TNF Receptors Expression --- p.126<br>Chapter 3.3.1.4.2 --- Effect of TNF-α Combination with A23187 on the Expression of TNF Receptors --- p.128<br>Chapter 3.3.1.4.3 --- Effects of Calcium Ionophore and Channel Blocker on TNF Receptors Expression --- p.130<br>Chapter 3.3.1.4.4 --- Effects of Calcium-Inducing Agents on TNF Receptors Gene Expressions --- p.130<br>Chapter 3.3.1.5 --- Effects of PKC Activator and Inhibitor on TNF-α- induced TNF Receptors Expressions --- p.135<br>Chapter 3.3.1.6 --- Effect of PKC and Ro31-8220 on IL-l-induced TNF Receptors Expressions --- p.138<br>Chapter 3.3.2 --- Expression of Calcium-sensing Receptor upon Different Drug Treatments --- p.138<br>Chapter 3.3.2.1 --- Effect of TNF-α on the Calcium-sensing Receptor Expression --- p.141<br>Chapter 3.3.2.2 --- Effects of Other Cytokines on CaSR Expression --- p.143<br>Chapter 3.3.2.3 --- Effect of A23187 on CaSR Expression --- p.143<br>Chapter 3.3.2.4 --- Effect of TNF-α and A23187 Combined Treatment on CaSR Expression --- p.146<br>Chapter 3.3.2.5 --- Effects of Calcium-inducing Agents on CaSR Expression --- p.149<br>Chapter 3.3.2.6 --- Effects of PKC Activator and PKC Inhibitor on CaSR Expression --- p.149<br>Chapter 3.3.3 --- Expression of PKC Isoforms upon Treatment with Different Drugs --- p.153<br>Chapter 3.3.3.1 --- Effect of TNF-α on the PKC Isoforms Expression --- p.155<br>Chapter 3.3.3.2 --- Effect of A23187 on the PKC Isoforms Expression --- p.155<br>Chapter 3.3.3.3 --- Effect of TNF-α and Calcium Ionophore Combined Treatment on PKC Isoforms Expression --- p.157<br>Chapter 3.3.3.4 --- Effects of Calcium-inducing Agents on PKC Isoforms Expression --- p.159<br>Chapter 3.3.4 --- Expression of the Transcription Factors c-fos and c-junin Drug Treatments --- p.161<br>Chapter 3.3.4.1 --- Effect of TNF-a on c-fos and c-jun Expression --- p.163<br>Chapter 3.3.4.2 --- Effect of A23187 on c-fos and c-jun Expression --- p.163<br>Chapter 3.3.4.3 --- Effect of TNF-a in Combination with A23187 on c- fos and c-jun Expression --- p.165<br>Chapter 3.3.4.4 --- Effects of Calcium-inducing Agents on c-fos and c- jun Expression --- p.167<br>Chapter 3.3.5 --- Effects of Different Drugs on Endogenous TNF-α Expression --- p.167<br>Chapter 3.3.5.1 --- Effect of TNF-α on Endogenous TNF-α Expression --- p.169<br>Chapter 3.3.5.2 --- Effect of A23187 on Endogenous TNF-α Expression --- p.169<br>Chapter 3.3.5.3 --- Effect of TNF-α in Combination with A23187 on the Expression of Endogenous TNF-α --- p.172<br>Chapter 3.3.5.4 --- Effects of Calcium-inducing Agents on Endogenous TNF-α Expression --- p.172<br>Chapter 3.3.6 --- Effect of Different Drugs on LL-1 Expression --- p.175<br>Chapter 3.3.6.1 --- Effect of TNF-a on IL-lα Expression --- p.177<br>Chapter 3.3.6.2 --- Effect of A23187 on the IL-lα Expression --- p.177<br>Chapter 3.3.6.3 --- Effect of Calcium Ionophore and TNF-α Combined Treatment on IL-1α Expression --- p.179<br>Chapter 3.3.6.4 --- Effects of Calcium-inducing Agents on IL-lα Expression --- p.179<br>Chapter 3.3.6.5 --- Effect of PKC Activator on the IL-1α Expression --- p.181<br>Chapter CHAPTER 4 --- DISCUSSIONS AND CONCLUSIONS<br>Chapter 4.1 --- "Effects of Cytokines, Ca2+ and PKC and Anti-TNF-α Antibodies on C6 Glioma Cells Proliferation" --- p.184<br>Chapter 4.2 --- The Role of Calcium in TNF-α-induced Signal Transduction Pathways --- p.186<br>Chapter 4.3 --- Gene Expressions in C6 Cells after TNF-a Stimulation --- p.187<br>Chapter 4.3.1 --- "Expression of TNF-α, TNF-Receptors and IL-1" --- p.187<br>Chapter 4.3.2 --- CaSR Expression --- p.190<br>Chapter 4.3.3 --- PKC Isoforms Expressions --- p.192<br>Chapter 4.3.4 --- Expression of c-fos and c-jun --- p.193<br>Chapter 4.4 --- Conclusion --- p.194<br>REFERENCES --- p.200

Les cellules épithéliales des voies aériennes respiratoires sécrètent du Cl- via le canal CFTR. La fibrose kystique est une maladie génétique fatale causée par des mutations de ce canal. La mutation la plus fréquente en Amérique du Nord, ∆F508, met en péril la maturation de la protéine et affecte les mécanismes d’activation du canal. Au cours des dernières années, plusieurs molécules ont été identifiées par criblage à haut débit qui peuvent rétablir l’activation de protéines CFTR mutées. Ces molécules sont nommées potentiateurs. Les canaux K+ basolatéraux, dont KCa3.1, jouent un rôle bien documenté dans l’établissement d’une force électromotrice favorable à la sécrétion de Cl- par CFTR dans les cellules épithéliales des voies aériennes respiratoires. Il a par exemple été démontré que l’application de 1-EBIO, un activateur de KCa3.1, sur des monocouches T84 résulte en une augmentation soutenue de la sécrétion de Cl- et que cette augmentation était réversible suite à l’application de CTX, un inhibiteur de KCa3.1(Devor et al., 1996). Dans le cadre d’une recherche de potentiateurs efficaces en conditions physiologiques et dans un contexte global de transport trans-cellulaire, il devient essentiel de considérer les effets des potentiateurs de CFTR sur KCa3.1. Une caractérisation électrophysiologique par la méthode du patch clamp et structurelle via l’utilisation de canaux modifiés par mutagenèse dirigée de différents potentiateurs de CFTR sur KCa3.1 fut donc entreprise afin de déterminer l’action de ces molécules sur l’activité de KCa3.1 et d’en établir les mécanismes. Nous présentons ici des résultats portant sur les effets sur KCa3.1 de quelques potentiateurs de CFTR possédant différentes structures. Un criblage des effets de ces molécules sur KCa3.1 a révélé que la genisteine, le SF-03, la curcumine et le VRT-532 ont des effets inhibiteurs sur KCa3.1. Nos résultats suggèrent que le SF-03 pourrait agir sur une protéine accessoire et avoir un effet indirect sur KCa3.1. La curcumine aurait aussi une action inhibitrice indirecte, probablement via la membrane cellulaire. Nos recherches sur les effets du VRT-532 ont montré que l’accessibilité au site d’action de cette v molécule est indépendante de l’état d’ouverture de KCa3.1. L’absence d’effets inhibiteurs de VRT-532 sur le mutant constitutivement actif V282G indique que cette molécule pourrait agir via l’interaction CaM-KCa3.1 et nécessiter la présence de Ca2+ pour agir. Par ailleurs, un autre potentiateur de CFTR, le CBIQ, a des effets potentiateurs sur KCa3.1. Nos résultats en canal unitaire indiquent qu’il déstabilise un état fermé du canal. Nos travaux montrent aussi que CBIQ augmente la probabilité d’ouverture de KCa3.1 en conditions sursaturantes de Ca2+, ainsi que son affinité apparente pour le Ca2+. Des expériences où CBIQ est appliqué en présence ou en absence de Ca2+ ont indiqué que l’accessibilité à son site d’action est indépendante de l’état d’ouverture de KCa3.1, mais que la présence de Ca2+ est nécessaire à son action. Ces résultats sont compatibles avec une action de CBIQ déstabilisant un état fermé du canal. Finalement, des expériences en Ba2+ nous ont permis d’investiguer la région du filtre de sélectivité de KCa3.1 lors de l’action de CBIQ et nos résultats pointent vers une action de CBIQ dans cette région. Sur la base de nos résultats nous concluons que CBIQ, un potentiateur de CFTR, aurait un effet activateur sur KCa3.1 via la déstabilisation d’un état fermé du canal à travers une action sur sa ‘gate’ au niveau du filtre de sélectivité. De plus, les potentiateurs de CFTR ayant montré des effets inhibiteurs sur KCa3.1 pourraient agir via la membrane ou via une protéine accessoire du canal ou sur l’interaction CaM-KCa3.1. Dans l’optique de traitements potentiels de la fibrose kystique, nos résultats indiquent que le CBIQ pourrait être un potentiateur efficace pusiqu’il est capable de trimuler à la fois KCa3.1 et CFTR. Par contre, dans les cas du VRT-532 et du SF-03, une inhibition de KCa3.1 pourraient en faire des potentiateurs moins efficaces.<br>Airway epithelial cells are the site of Cl- secretion through CFTR. Cystic fibrosis is a fatal genetic disease caused by mutations in CFTR. The most frequent mutation in North America (∆F508) results in impaired maturation and altered channel gating of the protein. In the last years, several small molecules were identified by high throughput screening that could restore mutated CFTR function. Compounds addressing CFTR gating defects are referred to as potentiators. The basolateral K+ channel KCa3.1 has been documented to play a prominent role in establishing a suitable driving force for CFTR-mediated Clsecretion in airway epithelial cells. It has been shown, for example, that the application of 1-EBIO on T84 monolayers results in a sustained increase of Clsecretion and that this current can be reversed by application of CTX, a KCa3.1 inhibitor (Devor et al., 1996). Thus, in a global approach of transepithelial transport, the research for physiologically relevant CFTR potentiators should also consider their effects on the KCa3.1 channel. Electrophysiological patch clamp measurements and channel structural modification by site directed mutagenesis were used to characterize the action of CFTR potentiators on KCa3.1 and study their molecular mode of action. In this work we present results on the effects on KCa3.1 of several CFTR potentiators of different structures. We observed that the CFTR potentiators genistein, curcumin, SF-03 and VRT-532 could inhibit KCa3.1 activity at concentrations known to activate CFTR. Our results suggest that SF- 03 could act indirectly on KCa3.1 through a mechanism involving an accessory protein. Curcumin would also have an indirect inhibitory effect, probably mediated by the plasma membrane, as documented for other ion channels. A detailed study of VRT-532 revealed that this molecule has access to its binding site in a state independent manner, and is poorly effective on the V282G mutant of KCa3.1, which is constitutively active. These results suggest that VRT-532 could act through the CaM/KCa3.1 complex and require the presence of Ca2+ to inhibit channel activity. In contrast, CBIQ, another CFTR potentiator, succeeded to activate KCa3.1. Our results in single channel show that CBIQ vii destabilizes a non conducting state of the channel. We also showed that this molecule increases the apparent Ca2+ affinity as well as the channel open probability, even in saturating Ca2+ conditions. Experiences in which Ba2+ was used as a probe were also performed to determine if the action mechanism of CBIQ involves an effect on the selectivity filter. Our results showed that Ba2+ could displace CBIQ from its interacting site, suggesting that the increases in channel activity induced by CBIQ could result from a change in the energetics of the channel at the level of the selectivity filter. On the basis of our results, we conclude that CBIQ, a CFTR potentiator, could activate KCa3.1 by destabilizing a non conducting state of the channel, probably through an action near the selectivity filter region. Also, CFTR potentiators having an inhibitory effect on KCa3.1 are likely to act through the plasmic membrane, the CaM/KCa3.1 interaction or an accessory protein of the channel. In a perspective of future treatments for CF, our results indicate that CBIQ could be an efficient potentiator since this product stimulates KCa3.1 as well as CFTR. Conversly, the VRT-532 and SF-03 could be less efficient than on CFTR alone, due to their inhibition of KCa3.1.