Investigations of pharmacologic properties of the renal CLC-K1 chloride channel co-expressed with barttin by the use of 2-(p-Chlorophenoxy)propionic acid derivatives and other structurally unrelated chloride channels blockers.

The role of voltage-gated chloride channels in the epileptogenesis of temporal lobe epilepsy

A two-electrode, voltage-clamp technique was used to measure the effect of the Cl(-) channel blockers, 9-anthracene carboxylic acid and niflumic acid, upon the ionic currents of oocytes of the South American toad Bufo arenarum. The main results were: (1) both blockers produced a reversible increase of the outward currents on a dose-dependent manner; (2) the activated outward current was voltage dependent; (3) the 9-anthracene carboxylic acid-sensitive current was blocked with barium; and (4) the effect of 9-anthracene carboxylic acid was more pronounced in a zero-K(+) solution than in standard (2 mmol l(-1)) or high (20 mmol l(-1)) K(+) solutions, indicating that a K(+) conductance is activated. The effect of the Cl(-) channel blockers could be due to a direct interaction with endogenous cationic channels. Another possible explanation is that Cl(-) that enter the cell during depolarizing steps in control solution inhibit this cationic conductance; thus, the blockade of Cl(-) channels by 9-anthracene carboxylic acid and niflumic acid would remove this inhibition, allowing the cationic current to flow freely.

The effects of monocarboxylic acid-derived Cl(-) channel blockers on cardiac depolarization-activated K(+) currents were investigated. Membrane currents in rat ventricular myocytes were recorded using the whole-cell configuration of the patch-clamp technique. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and niflumic acid (NFA) induced an outward current at 0 mV. Both NPPB and NFA failed to induce any current when used intracellularly or after K(+) in the bath and pipette solutions was replaced by equimolar Cs(+). Voltage pulse protocols revealed that NPPB and NFA enhanced the steady-state K(+) current but inhibited the transient outward K(+) current. Genistein, a tyrosine kinase (PTK) inhibitor, inhibited NPPB- and NFA-induced outward current. Another PTK inhibitor, lavendustin A, produced a comparable effect. In contrast, the inactive analogue of genistein, daidzein, was ineffective. Orthovanadate, a tyrosine phosphatase inhibitor, markedly slowed the deactivation of the outward current induced by NPPB and NFA. The protein kinase A (PKA) inhibitor H-89 inhibited NPPB-induced outward current at 0 mV. In contrast, the protein kinase C (PKC) inhibitor H-7 was without significant effect on the action of NPPB. Pretreatment of the myocytes with genistein or H-89 prevented the enhancing effect of NPPB. Increasing intracellular Cl(-) from 22 to 132 mm slightly reduced NPPB-induced outward current at 0 mV. These results demonstrate that the monocarboxylic acid-derived Cl(-) channel blockers NPPB and NFA enhance cardiac steady-state K(+) current, and suggest that the enhancing effect of the Cl(-) channel blockers is mediated by stimulation of PKA and PTK signalling pathways.

Effects of several Cl(-) channel blockers on ionic currents in mouse embryos were studied using whole-cell patch-clamp and microelectrode methods. Microelectrode measurements showed that the resting membrane potential of early embryonic cells (1-cell stage) was -23 mV and that reduction of extracellular Cl(-) concentration depolarized the membrane, suggesting that Cl(-) conductance is a major contributor for establishing the resting membrane potential. Membrane currents recorded by whole-cell voltage clamp showed outward rectification and confirmed that a major component of these embryonic currents are carried by Cl(-) ions. A Cl(-) channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), suppressed the outward rectifier current in a voltage- and concentration-dependent manner. Other Cl(-) channel blockers (5-nitro-2-[3-phenylpropyl-amino] benzoic acid and 2-[3-(trifluoromethyl)-anilino] nicotinic acid [niflumic acid]) similarly inhibited this current. Simultaneous application of niflumic acid with DIDS further suppressed the outward rectifier current. Under high osmotic condition, niflumic acid, but not DIDS, inhibited the Cl(-)current, suggesting the presence of two types of Cl(-) channels: a DIDS-sensitive (swelling-activated) channel, and a DIDS-insensitive (niflumic acid-sensitive) Cl(-) channel. Anion permeability of the DIDS-insensitive Cl(-) current differed from that of the compound Cl(-) current: Rank order of anion permeability of the DIDS-sensitive Cl(-) channels was I(-) = Br(-) > Cl(-) > gluconate(-), whereas that of the DIDS-insensitive Cl(-) channel was I(-) = Br(-) > Cl(-) >> gluconate(-). These results indicate that early mouse embryos have a Cl(-) channel that is highly permeable to amino acids, which may regulate intracellular amino acid concentration.

CLC-K Cl(-) channels belong to the CLC protein family. In kidney and inner ear, they are involved in transepithelial salt transport. Mutations in ClC-Kb lead to Bartter's syndrome, and mutations in the associated subunit barttin produce Bartter's syndrome and deafness. We have previously found that 3-phenyl-CPP blocks hClC-Ka and rClC-K1 from the extracellular side in the pore entrance. Recently, we have shown that niflumic acid (NFA), a nonsteroidal anti-inflammatory fenamate, produces biphasic behavior on human CLC-K channels that suggests the presence of two functionally different binding sites: an activating site and a blocking site. Here, we investigate in more detail the interaction of NFA on CLC-K channels. Mutants that altered block by 3-phenyl-2-(p-chlorophenoxy)propionic acid (CPP) had no effect on NFA block, indicating that the inhibition binding site of NFA is different from that of 3-phenyl-CPP and flufenamic acid. Moreover, NFA does not compete with extracellular Cl(-) ions, suggesting that the binding sites of NFA are not located deep in the pore. Differently from ClC-Ka, on the rat homologue ClC-K1, NFA has only an inhibitory effect. We developed a quantitative model to describe the complex action of NFA on ClC-Ka. The model predicts that ClC-Ka possesses two NFA binding sites: when only one site is occupied, NFA increases ClC-Ka currents, whereas the occupation of both binding sites leads to channel block.

High-performance liquid chromatographic determination of citalopram and four of its metabolites in plasma and urine samples from psychiatric patients

A new semifluorinated sulfonated copolytriazole is synthesized, maintaining a particular degree of sulfonation of 80% following the click reaction of two terminal azides 1,4‐bis‐[{2′‐trifluromethyl 4′‐(4″‐azidophenyl)phenoxy}]phenyl, and 4,4′‐diazido‐2,2′‐stilbenedisulfonic acid disodium salt with equal molar amounts of two dialkyne monomers namely, 1,3‐diethynylbenzene and 3,5‐bis(prop‐2‐ynyloxy)benzoic acid and designated as PTEHCSH‐80. Further, the copolytriazole is used to prepare polymer composites employing the sol‐gel reaction of the starting precursor sol 3‐(trihydroxylsilyl) propane‐1‐sulfonic acid. The weight percentage (wt%) of the filler is varied from 0 to 10 wt%, and a series of polymer composite films are prepared and abbreviated as PTEHCSH‐80/X (where X is wt% of the filler, X = 0. 2.5, 5.0, 7.5, 10.0). All the polymer composites are characterized through FTIR spectroscopic techniques. The composite films revealed various desirable properties such as high thermochemical and dimensional and mechanical stabilities in their corresponding acid form. Moreover, the composites exhibited proton conductivities ranging from 62 to 98, 117 to 182, and 131 to 195 mS/cm at three different temperatures 30°C, 80°C, and 90°C, respectively, in a hydrated state.

New crosslinked sulfonated polytriazoles: Proton exchange properties and microbial fuel cell performance

The direct electrophysiological characterization of sperm Ca(2+) channels has been precluded by their small size and flat shape. An alternative to study these channels is to use spermatogenic cells, the progenitors of sperm, which are larger and easier to patch-clamp. In mouse and rat, the only voltage-dependent Ca(2+) currents displayed by these cells are of the T type. Because compounds that block these currents inhibit the zona pellucida-induced Ca(2+) uptake and the sperm acrosome reaction (AR) at similar concentrations, it is likely that they are fundamental for this process. Recent single channel recordings in mouse sperm demonstrated the presence of a Cl(-) channel. This channel and the zona pellucida (ZP)-induced AR were inhibited by niflumic acid (NA), an anion channel blocker [Espinosa et al. (1998): FEBS Lett 426:47-51]. Because NA and other anion channel blockers modulate cationic channels as well, it became important to determine whether they affect the T-type Ca(2+) currents of spermatogenic cells. These currents were blocked in a voltage-dependent manner by NA, 1, 9-dideoxyforskolin (DDF), and 5-nitro-2-(3-phenylpropylamine)benzoic acid (NPPB). The IC(50) values at -20 mV were 43 microM for NA, 28 microM for DDF, and 15 microM for NPPB. Moreover, DDF partially inhibited the ZP-induced AR (40% at 1 microM) and NPPB displayed an IC(50) value of 6 microM for this reaction. These results suggest that NA and DDF do not inhibit the ZP-induced AR by blocking T-type Ca(2+) currents, while NPPB may do so. Interestingly 200 microM NA was basically unable to inhibit alpha1E Ca(2+) channels expressed in Xenopus oocytes, questioning that this alpha subunit codes for the T-type Ca(2+) channels present in spermatogenic cells. Evidence for the presence of alpha1C, alpha1G, and alpha1H in mouse pachytene spematocytes and in round and condensing spermatids is presented.

Results from a relatively small case-control study recently showed that niflumic acid increases the risk of serious mucocutaneous reactions in children. As a consequence, the Italian Ministry of Health sent a "Dear Doctor" letter in June 2001 to warn pediatricians about the alleged adverse effects. The objective of this study was to estimate and compare the incidence of mild and severe mucocutaneous reactions among children using niflumic acid, other nonsteroidal antiinflammatory drugs (NSAIDs), or nonopioid analgesics. Retrospective cohort study. Italy is one of the few countries in which a specific primary care system is devoted to children up to 14 years of age: every child is registered at birth and receives free medical care from 1 of the approximately 6000 family pediatricians working for the National Health Service. This study was conducted with the Pedianet network of Italian family pediatricians who use computerized electronic patient records for routine care; 185 pediatricians participated in the study. The patient records comprise information on demographics, diagnoses, symptoms, prescriptions, referrals, laboratory examinations, and hospitalizations. Children aged 0 to 14 years and registered with 1 of the collaborating pediatricians between January 1, 1998, and May 31, 2001. The incidence rate of severe (hospitalized or referred) and mild mucocutaneous reactions (exanthema, disseminated or localized pruritus, urticaria, angioedema, fixed eruption, dermatitis, erythema multiforme, vesicles, bullae, pustules, toxic epidermal necrolysis, purpura, and vasculitis) was estimated during use of niflumic acid, other NSAIDs, or nonopioid analgesics. For each episode of drug use, the following covariates were assessed: age, gender, region, year, indication for study drug, use of antibiotics, antimycotic agents, glucocorticoids, and other NSAIDs. Multivariate Poisson regression analysis was used to estimate the adjusted relative risk of mucocutaneous disorders during use of niflumic acid compared with use of other NSAIDs or use of acetaminophen alone. The population included 193727 children, 45351 of whom received at least 1 of the study drugs. The most frequently prescribed drugs were niflumic acid, acetaminophen, and propionic acid derivatives (ketoprofen and flurbiprofen). Users of niflumic acid (n = 32150) were younger and slightly more often had otitis media or upper respiratory tract infections as an indication compared with the other NSAIDs. During use of the various study drugs we identified 1451 mild mucocutaneous events and 42 severe reactions. The incidence rates of severe and mild mucocutaneous reactions after the administration of any study drug were 10.3 per 100000 exposure person-days and 3.7 per 1000 exposure person-days, respectively. Both incidence rates decreased strongly with increasing age. In comparison with other NSAIDs, the adjusted relative risks of niflumic acid were 0.5 (95% confidence interval: 0.23-1.27) for severe and 0.9 (95% confidence interval: 0.79-1.11) for mild mucocutaneous reactions. The use of acetaminophen as a reference category instead of other NSAIDs, restriction of the children to those who received NSAIDs for respiratory tract infections, or restriction to those who did not use antibiotics never revealed an increased risk of serious or mild mucocutaneous reactions during use of niflumic acid. In comparison with other NSAIDs or acetaminophen, niflumic acid is not associated with an increased risk of severe or mild mucocutaneous reactions in children. This was true for the different age groups and various types of mucocutaneous reactions, was independent of the concomitant use of antibiotics, and was not sensitive to changes in our assumptions regarding exposure and outcomes.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.

Adenosine triphosphate (ATP) plays the role of an autocrine/paracrine signal molecule in a variety of cells. So far, however, the membrane machinery in the export of intracellular ATP remains poorly understood. Activation of B2-receptor with bradykinin-induced massive release of ATP from cultured taenia coli smooth muscle cells. The evoked release of ATP was unaffected by gap junction hemichannel blockers, such as 18alpha-glycyrrhetinic acid and Gap 26. Furthermore, the cystic fibrosis transmembrane regulator (CFTR) coupled Cl(-) channel blockers, CFTR(inh)172, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, Gd3(+) and glibenclamide, failed to suppress the export of ATP by bradykinin. On the other, the evoked release of ATP was greatly reduced by multidrug resistance protein (MRP) transporter inhibitors, MK-571, indomethacin, and benzbromarone. From western blotting analysis, blots of MRP 1 protein only, but not MRP 2 and MRP 3 protein, appeared at 190 kD. However, the MRP 1 protein expression was not enhanced after loading with 1 muM bradykinin for 5 min. Likewise, niflumic acid and fulfenamic acid, Ca2(+)-activated Cl(-) channel blockers, largely abated the evoked release of ATP. The possibility that the MRP transporter system couples with Ca2(+)-activated Cl(-) channel activities is discussed here. These findings suggest that MRP transporters, probably MRP 1, unlike CFTR-Cl(-) channels and gap junction hemichannels, may contribute as membrane machinery to the export of ATP induced by G-protein-coupled receptor stimulation.

Naproxen is a non-steroidal anti-inflammatory drug (NSAID), belonging to propionic acid group, and its chemical structure is a 6-metoxi-metil-2-naftalenoacetic acid. Fixed drug eruptions (FDE) have been rarely reported. A 38-year-old woman referred that after 2 hours of taking 2 tablets of naproxen for a headache, she developed several edematous and dusky-red macules, one on right forearm and the other two in both thighs and she was diagnosed with FDE probably due to naproxen. We performed patch testing (PT) (Nonweven Patch Test Strips Curatest® Lohman & Rauscher International, Rangsdorf, Germany), with ibuprofen (5% Petrolatum), ketoprofen (2.5% Petrolatum), naproxen and nabumetone (both 10% in DMSO) on the residual lesion of the forearm with naproxen and in both thighs with ibuprofen, ketoprofen and nabumetone. Readings at day 1 (D1) and day 2 (D2) showed negative results to ibuprofen, ketoprofen and nabumetone, but were positive to naproxen in D1. A single blind oral challenge test (SBOCT) with other propionic acid derivates were performed in order to check for crossreactivity between them: ibuprofen, ketoprofen and nabumetone were administered and all drugs were well tolerated. In our patient PT confirmed the diagnosis and allowed us to study the cross-reactivity between NSAIDs of the same group, and confirmed by SBOCT. Cross-reactivity between propionic acid derivatives was studied. This is a case of hypersensitivity to naproxen with good tolerance to other propionic acids NSAIDs (ibuprofen and ketoprofen) and nabumetone, confirmed by PT and SBOCT. Some relavent patents for fixed drug eruption are discussed.

Fluoroquinolones and propionic acid derivatives are widely used antibacterials and non-steroidal anti-inflammatory drugs, respectively, which have been reported to frequently trigger drug hypersensitivity reactions. Such reactions are induced by inflammatory mediators such as cytokines and chemokines. The present study investigated whether levofloxacin, a fluoroquinolone, and loxoprofen, a propionic acid derivative, have the potential to induce immune-related gene expression in dendritic cell-like cell lines such as HL-60, K562, and THP-1, and immortalized keratinocytes such as HaCaT. The expression of IL-8, MCP-1, and TNFα messenger RNA (mRNA) was found to increase following treatment with levofloxacin or loxoprofen in HL-60 cells. In addition, these drugs increased the mRNA content of annexin A1, a factor related to keratinocyte necroptosis in patients with severe cutaneous adverse reactions. Inhibition studies using specific inhibitors of mitogen-activated protein (MAP) kinases and NF-κB suggest that the extracellular signal-regulated kinase (ERK) pathway is the pathway principally involved in the induction of cytokines and annexin A1 by levofloxacin, whereas the involvement of MAP kinases and NF-κB in the loxoprofen-induced gene expression of these factors may be limited. Fluoroquinolones and propionic acid derivatives that are structurally related to levofloxacin and loxoprofen, respectively, were also found to induce immune-related gene expression in HL-60 cells. Collectively, these results suggest that fluoroquinolones and propionic acid derivatives have the potential to induce the expression of immune-related factors and that an in vitro cell-based assay system to detect the immune-stimulating potential of systemic drugs might be useful for assessing the risk of drug hypersensitivity reactions.

Determination of naturally derived propionic, benzoic, and sorbic acids in seafood, meats, and fruits during storage