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PubMed2005

Neuronal chloride accumulation in olfactory epithelium of mice lacking NKCC1.

When stimulated with odorants, olfactory receptor neurons (ORNs) produce a depolarizing receptor current. In isolated ORNs, much of this current is caused by an efflux of Cl-. This implies that the neurons have one or more mechanisms for accumulating cytoplasmic Cl- at rest. Whether odors activate an efflux of Cl- in intact olfactory epithelium, where the ionic environment is poorly characterized, has not been previously determined. In mouse olfactory epithelium, we found that >80% of the summated electrical response to odors is blocked by niflumic acid or flufenamic acid, each of which inhibits Ca2+-activated Cl- channels in ORNs. This indicates that ORNs accumulate Cl- in situ. Recent evidence has shown that NKCC1, a Na+-K+-2Cl- cotransporter, contributes to Cl- accumulation in mammalian ORNs. However, we find that the epithelial response to odors is only reduced by 39% in mice carrying a null mutation in Nkcc1. As in the wild-type, most of the response is blocked by niflumic acid or flufenamic acid, indicating that the underlying current is carried by Cl-. We conclude that ORNs effectively accumulate Cl- in situ even in the absence of NKCC1. The Cl- -transport mechanism underlying this accumulation has not yet been identified.

Animals↗

PubMed1995

Alpha-adrenergic stimulation activates a calcium-sensitive chloride current in brown fat cells.

The first response of brown adipocytes to adrenergic stimulation is a rapid depolarizing conductance increase mediated by alpha-adrenergic receptors. We used patch recording techniques on cultured brown fat cells from neonatal rats to characterize this conductance. Measurements in perforated patch clamped cells showed that fast depolarizing responses were frequent in cells maintained in culture for 1 d or less, but were seen less often in cells cultured for longer periods. Ion substitution showed that the depolarization was due to a selective increase in membrane chloride permeability. The reversal potential for the depolarizing current in perforated patch clamped cells indicated that intracellular chloride concentrations were significantly higher than expected if chloride were passively distributed. The chloride conductance could be activated by increases in intracellular calcium, either by exposing intact cells to the ionophore A23187 or by using pipette solutions with free calcium levels of 0.2-1.0 microM in whole-cell configuration. The chloride conductance did not increase monotonically with increases in intracellular calcium, and going whole cell with pipette-free calcium concentrations > or = 10 microM rapidly inactivated the current. The chloride currents ran down in whole-cell recordings using intracellular solutions of various compositions, and were absent in excised patches. These findings imply that cytoplasmic factors in addition to intracellular calcium are involved in regulation of the chloride conductance. The chloride currents could be blocked by niflumic acid or flufenamic acid with IC50s of 3 and 7 microM, or by higher concentrations of SITS (IC50 = 170 microM), DIDS (IC50 = 50 microM), or 9-anthracene carboxylic acid (IC50 = 80 microM). The chloride conductance activated in whole cell by intracellular calcium had the permeability sequence PNOS > PI > PBr > PCl >> Paspartate, measured from either reversal potentials or conductances. Instantaneous current-voltage relations for the calcium-activated chloride currents were linear in symmetric chloride solutions. Much of the current was time and voltage independent and active at all membrane potentials between -100 and +100 mV, but an additional component of variable amplitude showed time-dependent activation with depolarization. Volume-sensitive chloride currents were also present in brown fat cells, but differed from the calcium-activated currents in that they responded to cell swelling, required intracellular ATP in whole-cell recordings, showed no sensitivity to intracellular or extracellular calcium levels, and were relatively resistant to block by niflumic and flufenamic acids. (ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue, Brown↗

PubMed2000

Fenamates and the potent inhibition of human liver phenol sulphotransferase.

1. The inhibition of the human liver phenol sulphotransferase (HL-PST) and catechol sulphotransferase (HL-CST) by five fenamates has been studied and the activities of HL-PST and HL-CST were measured with 4-nitrophenol and dopamine as substrates, respectively. 2. The IC50 for inhibition of HL-PST were 0.02 microM (mefenamic acid); 0.12 microM (tolfenamic acid); 0.28 microM (niflumic acid); 0.87 microM (meclofenamic acid) and 1.50 microM (flufenamic acid). 3. HL-CST was less susceptible than HL-PST to the inhibition by fenamates and the IC50 for HL-CST were 36 microM (tolfenamic acid); 70 microM (flufenamic acid); 76 microM (mefenamic acid); 180 microM (niflumic acid) and 185 microM (meclofenamic acid). 4. The ratios of the IC50 for HL-CST:HL-PST were drug-dependent and ranged from 47 (flufenamic acid) to 3800 (mefenamic acid). Mefenamic acid is a relatively potent and selective inhibitor of HL-PST. 5. The IC50 for HL-PST obtained with mefenamic acid was three orders of magnitude lower than the peak plasma concentration of this drug after an oral dose of 0.5 g. Accordingly, mefenamic acid should impair sulphation in vivo.

Arylsulfotransferase↗

PubMed1987

[The mode of anti-inflammatory action of a topical non-steroidal anti-inflammatory drug, etofenamate].

In order to ascertain the mode of anti-inflammatory action of a topical non-steroidal anti-inflammatory drug, etofenamate which is a diethylene glycol ester of flufenamic acid, the in vitro test for the mechanism of the action were carried out. Etofenamate (3 microM) was hydrolysed to flufenamic acid at a rate of 39.5% and 57.0% of the dose during 30 and 60 min incubation, respectively, when incubated with rat peritoneal macrophages stimulated with starch and bacto peptone in phosphate-buffered saline. PGE2 generation by these cells in MEM medium was dose-relatedly inhibited with etofenamate as well as flufenamic acid at the dosage range of 1 to 30 microM. This suggests that unchanged etofenamate is active, since the highest conversion rate of etofenamate to flufenamic acid was 15% of the dose during the incubation. Etofenamate produced a dose-related inhibition against lipoxygenase prepared from peritoneal polymorphonuclear leucocytes of guinea pigs, and its activity (IC50 = 5.3 X 10(-5) M) was stronger than that of caffeic acid; flufenamic acid was inactive. Inhibitory activity of etofenamate was one-third or less that of flufenamic acid against the hypotonic-hyperthermic lysis of rat erythrocytes and heat-denaturation of bovine serum albumin. From these results, it was suggested that topically applied etofenamate produces its anti-inflammatory action through prostaglandin synthesis inhibition by flufenamic acid produced in the inflammatory tissue and inhibition of prostaglandin synthesis by macrophages and lipoxygenase inhibition by unchanged etofenamate.

Administration, Topical↗

PubMed2003

Diclofenac inhibition of sodium currents in rat dorsal root ganglion neurons.

The effects of diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), on the fast tetrodotoxin-sensitive (TTX-S) and the slow tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion neurons were investigated using the whole-cell patch-clamp method. Diclofenac suppressed both sodium currents in a dose-dependent manner. The apparent dissociation constants for the diclofenac suppression of TTX-S and TTX-R sodium currents were estimated to be 14 and 97 microM, respectively, at a holding potential of -80 mV. Diclofenac had no effect on the kinetic parameters of the activation process in either type of sodium current. However, diclofenac produced shifts of the steady-state inactivation curves in the hyperpolarizing direction in both types of sodium currents in a dose-dependent manner. At sufficiently negative holding potentials, the inhibitory effects of diclofenac on both types of sodium currents were minimal. The results suggested that diclofenac might bind to sodium channels with a greater affinity when they are in the inactivated state than when they are in the resting state. Effects of other NSAIDs (acetylsalicylic acid, antipyrin, indomethacin and flufenamic acid) on sodium currents were tested. Among these, only flufenamic acid suppressed the sodium currents to a considerable extent. Thus, the chemical structure of each NSAID, not the inhibition of cyclooxygenase, seems to be an important determinant in the sodium current inhibition. The suppression of sodium currents in sensory neurons by diclofenac and flufenamic acid would contribute to their analgesic activity in addition to their inhibition of cyclooxygenase.

Animals↗

PubMed1998

Involvement of stretch-activated Cl- channels in ramification of murine microglia.

A stretch-activated Cl- current (ICl) was investigated in cultured murine microglia using the whole-cell configuration of the patch-clamp technique. After application of membrane stretch, a Cl- current appeared within seconds, and its amplitude increased further within 3-8 min. ICl underwent rundown, which was prevented by addition of 4 mM ATP to the intracellular perfusing solution. The stretch-activated Cl- current exhibited outward rectification and did not show any voltage-dependent gating. Lowering the concentration of extracellular Cl- from 142 to 12 mM by equimolar substitution of Cl- with gluconate shifted the reversal potential of ICl by 41.6 +/- 1.8 mV in the depolarizing direction. 4, 4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) blocked ICl in a voltage- and time-dependent manner. At a test potential of +40 mV, a half-maximal blockade at 16.1 microM DIDS and at 71.0 microM SITS was determined for ICl. At a concentration of 200 microM, 5-nitro-2-(3-phenylpropylamino)benzoic acid or flufenamic acid blocked ICl by 88% and 75%, respectively. Each of these four Cl- channel blockers reversibly inhibited the ramification process of microglia, whereas blockers of voltage-gated Na+ and K+ channels did not affect the transformation of microglia from their ameboid into the ramified phenotype. It is suggested that in microglia functional stretch-activated Cl- channels are required for the induction of ramification but not for maintaining the ramified shape.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2000

Simultaneous analysis of anthranilic acid derivatives in pharmaceuticals and human urine by high-performance liquid chromatography with isocratic elution.

A high-performance liquid chromatographic (HPLC) method for simultaneous determination of mefenamic acid (MFA), flufenamic acid (FFA) and tolfenamic acid (TFA) is presented for application to pharmaceuticals and human urine. Isocratic reversed-phase HPLC was employed for quantitative analysis using tetra-pentylammonium bromide (TPAB) as an ion-pair reagent. Urine samples were purified by solid-phase extraction using a silica-based strong anion-exchanger, Bond-Elut SAX cartridge. The HPLC assay was carried out using a Wakosil ODS 5C18 column (5 microm, 150x4.6 mm I.D.). The mobile phase consisted of 1.9 g of TPAB dissolved in 1:1 of a mixture of acetic acid-sodium acetate buffer solution, pH 5.0, and acetonitrile (11:9, v/v). The calibration curves of MFA, FFA and TFA showed good linearity in the concentration range of 33-167 microg/ml with a wavelength of 280 nm for pharmaceuticals, and in the low concentration range (1.7-30.1 microg/ml) with a wavelength of 230 nm for biological fluids. The correlation coefficients were better than 0.9999 in all cases. The lower limits of detection (defined as a signal-to-noise ratio of about 3) were approximately 2 ng for MFA, 3.5 ng for FFA and 2.5 ng for TFA. The procedure described here is rapid, simple, selective and is suitable for routine analysis of pharmaceuticals and pharmacokinetic studies in human urine samples.

Calibration↗

PubMed2005

Comparison of functional properties of the Ca2+-activated cation channels TRPM4 and TRPM5 from mice.

Non-selective cation (NSC) channels activated by intracellular Ca2+ ([Ca2+]i) play an important role in Ca2+ signaling and membrane excitability in many cell types. TRPM4 and TRPM5, two Ca2+-activated cation channels of the TRP superfamily, are potential molecular correlates of NSC channels. We compared the functional properties of mouse TRPM4 and TRPM5 heterologously expressed in HEK 293 cells. Dialyzing cells with different Ca2+ concentrations revealed a difference in Ca2+ sensitivity between TRPM4 and TRPM5, with EC50 values of 20.2+/-4.0 microM and 0.70+/-0.1 microM, respectively. Similarly, TRPM5 activated at lower Ca2+ concentration than TRPM4 when [Ca2+]i was raised by UV uncaging of the Ca2+-cage DMNP-EDTA. Current amplitudes of TRPM4 and TRPM5 were not correlated to the rate of changes in [Ca2+]i. The Ca2+ sensitivity of both channels was strongly reduced in inside-out patches, resulting in approximately 10-30 times higher EC50 values than under whole-cell conditions. Currents through TRPM4 and TRPM5 deactivated at negative and activated at positive potentials with similar kinetics. Both channels were equally sensitive to block by intracellular spermine. TRPM4 displayed a 10-fold higher affinity for block by flufenamic acid. Importantly, ATP4- blocked TRPM4 with high affinity (IC50 of 0.8+/-0.1 microM), whereas TRPM5 is insensitive to ATP4- at concentrations up to 1 mM.

3T3 Cells↗

PubMed2000

Ca2+ influx mediates apoptosis induced by 4-aminopyridine, a K+ channel blocker, in HepG2 human hepatoblastoma cells.

Apoptosis appears to be implicated in the pathogenesis and therapeutic applications of cancer. In this study we investigated the induction of apoptosis by 4-aminopyridine (4-AP), a K(+) channel blocker, and its mechanism in HepG2 human hepatoblastoma cells. 4-AP reduced cell viability and induced DNA fragmentation, a hallmark of apoptosis, in a dose-dependent manner. In addition, 4-AP induced a sustained increase in intracellular Ca(2+) concentration, which was completely inhibited by the extracellular Ca(2+) chelation with EGTA. 4-AP also induced Mn(2+) influx, indicating that the 4-AP-induced increased intracellular Ca(2+) levels were due to activation of Ca(2+) influx pathway. 4-AP also depolarized membrane potential that was measured by using di-O-C(5)(3), a voltage-sensitive fluorescent dye. 4-AP-induced Ca(2+) influx was significantly inhibited not by voltage-operative Ca(2+) channel blockers (nifedipine or verapamil), but by flufenamic acid (FA), a known nonselective cation channel blocker. Quantitative analysis of apoptosis by the flow cytometry revealed that treatment with either FA or BAPTA, an intracellular Ca(2+) chelator, significantly inhibited the 4-AP-induced apoptosis. Taken together, these results suggest that the observed 4-AP-induced apoptosis in the HepG2 cells may result from Ca(2+) influx through the activation of voltage-sensitive Ca(2+)-permeable non-selective cation channels. These results further suggest that membrane potential change by modulation of K(+) channel activity may be involved in the mechanism of apoptosis in human hepatoma cells.

4-Aminopyridine↗

PubMed1981

[Metabolism of etofenamate / Identification and analytic of metabolites, their pharmacological properties and species dependence of metabolism (author's transl)].

After oral application of etofenamate to animals (rat, rabbit, dog, monkey) unchanged etofenamate and numerous metabolites are found in urine. Analytical properties (thin-layer chromatographic behavior, UV- and fluorescence data) of etofenamate, 5-hydroxy-, 4'-hydroxy, 5,4'-dihydroxy-etofenamate, flufenamic acid, 5-hydroxy-, 4'-hydroxy-, 5,4'-dihydroxy-flufenamic acid are described. Derivatives of etofenamate and flufenamic acid are excreted by rabbit, dog, monkey and man, whereas flufenamic acid derivatives are excreted preferentially by rats; profound degradation takes place in dogs. Metabolism in man is more similar to monkey than to dog and rodents. Metabolic pattern after oral and cutaneous application is quite similar. The six hydroxy derivatives have no pharmacological activity--they do not contribute to the pharmacological action of the substance.

Animals↗

PubMed1990

[Plasma level studies in volunteers after intramuscular injection of various doses of etofenamate in an oily solution].

Plasma level Studies on Volunteers after Intramuscular Application of Different Doses of Etofenamate in Oily Solution. After i.m. injections of etofenamate (active substance of Rheumon i.m.) in oily solution to 12 volunteers, courses of plasma levels of etofenamate, flufenamic acid and fenamate (sum of etofenamate and flufenamic acid) were measured by HPTLC. Maximum levels of etofenamate, flufenamic acid and fenamate, as well as areas under the plasma level time curve (AUC) after 250, 500 and 1000 mg etofenamate respectively are proportional to dose. Maxima of fenamate plasma levels are reached after 6.3, 6.2 and 5.4 h respectively, half maximal levels are present already after 2 h. The mean residence time is 21.8, 18.8 and 15.7 h. These values obtained from different doses are not statistically different from each other. Pharmacokinetics are therefore linear and dose independent. The courses of fenamate levels can be described by a two compartment model. The elimination half lives after 250, 500 and 1000 mg are 2.1, 2.3 and 1.9 h, the invasion half-lives (dominant half-life) 8.8, 7.8 and 6.8 h. Terminal half-lives are 50.3, 63.7 and 35.4 h. Since plasma levels have decreased to 2% of the maximum level after one terminal half-life, they have no practical importance for the duration of activity or for accumulation. No sex related differences are found for dose dependent and independent parameters. From the data it can be derived that after i.m. injection of etofenamate in oily solution a prolongation of the dominant half-life occurs by a factor of 4-5 (as compared to oral data) which is caused by prolonged liberation from the oily depot. This long lasting liberation of etofenamate leads to a prolonged residence time after a fast increase, at the same time avoiding unnecessary high peak levels. Therefore it is guaranteed that even after i.m. administration of 1000 mg etofenamate in oily solution plasma levels of fenamate do not exceed those after 300 mg given orally. According to pharmacokinetic data a fast onset of action, good tolerability and a therapeutic action over a period of 24 h can be expected.

Adult↗

PubMed1990

[Animal experimental evidence of the long-lasting efficacy of etofenamate by prolongation of the half-life after intramuscular application].

Animal Experimental Evidence of Long-lasting Liberation of Etofenamate by Half-life Prolongation after Intramuscular Application. The purpose of this investigation was to show in animal experiments that by i.m. injection of etofenamate (active substance of Rheumon i.m.) in oily solution the following effects could be obtained: a fast onset of action (gain of therapeutically relevant drug levels shortly after injection) a long-lasting efficacy (prolonged liberation from the oil depot) and better tolerability as compared to other intramuscularly applicable antiinflammatory drugs (avoidance of high plasma spikes). Etofenamate in rats is liberated with a half-life of 1.29 days from the place of application (cutaneous half-life 8.5 h). Flufenamic acid in muscles is found only in traces. After i.m. administration of etofenamate to dogs maximum plasma levels of etofenamate and flufenamic acid were reached within 2 and 4 h, resp. The mean half-lives of plasma elimination are 14 h for etofenamate and 23.2 h for flufenamic acid formed esterolytically from etofenamate (flufenamic acid oral half-life 2-4 h). Maximum plasma levels after etofenamate are only 6.5-11.8% of the maximum levels after equivalent amounts of flufenamic acid administered orally. According to these data etofenamate i.m. is a drug formulation with fast increasing plasma levels, prolonged half-life and lower maximum plasma levels as compared to orally administered preparations. The results are confirmed in animals (pharmacodynamics, toxicology and tolerability) and man (kinetics, clinical studies).

Administration, Oral↗

PubMed1999

Enhancement of low density lipoprotein catabolism by non-steroidal anti-inflammatory drugs in cultured HepG2 cells.

Several clinical studies have shown that different types of non-steroidal anti-inflammatory drugs (NSAIDs) can reduce the cholesterol content of atherosclerotic blood vessels. The mechanism of this reduction is not established. One possibility is that NSAIDs affect low density lipoprotein (LDL) catabolism. In this study, we investigated the effect of the NSAIDs, indomethacin, flufenamic acid, ibuprofen, acetaminophen, and also acetylsalicylic acid on LDL binding, cell-association and degradation in cultured hepatoma HepG2 cells. LDL was labelled with 125I to study LDL catabolism. Furthermore, dextran sulphate, a substance that is known to release bound LDL from its receptors, was used to study LDL receptor activity. Reverse transcription-polymerase chain reaction was used to study the messenger RNA (mRNA) of LDL receptor. Our results show that flufenamic acid, indomethacin, and to a lesser extent ibuprofen, and acetaminophen increase LDL binding, cell-association, and degradation. Flufenamic acid was most potent and increased LDL catabolism by 50-70%, whereas acetylsalicylic acid had only a modest effect. Also, flufenamic acid and indomethacin were both found to increase the synthesis of mRNA of the LDL receptor with a subsequent increase of LDL receptor protein. We also investigated the effect of indomethacin on LDL binding in the presence of the 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase inhibitor, fluvastatin. We found that both indomethacin and fluvastatin had an additive up-regulatory effect on LDL receptor activity. In addition the effect of flufenamic acid on cell-associated LDL was examined in the presence of cyclosporine, which is known to decrease LDL catabolism. The results show that flufenamic acid can restore the inhibitory effect of cyclosporine. The study thus shows that NSAIDs enhance LDL catabolism due to increased synthesis of the mRNA for LDL receptor protein. This action might contribute to the lipid-lowering effect of NSAIDs.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1990

[Renal elimination and metabolism of etofenamate in volunteers after administration of various doses].

Renal Elimination and Metabolism of Etofenamate after Intramuscular Administration of Different Doses to volunteers. Renal elimination of etofenamate (active substance of Rheumon i.m.) after i.m. injection of oily solution of etofenamate to volunteers was investigated by HPTLC and GC. After injection of 250, 500 and 1000 mg etofenamate, free and conjugated flufenamic acid (flu), 5-hydroxy- and 4'-hydroxy flufenamic acid (5-OH-flu, 4'-OH-flu) were found as main metabolites in urine. Besides that several minor metabolites were identified. The ratio of free to conjugated metabolites was 1:10 to 1:25. From the doses administered 30% were eliminated as main metabolites. Overall amounts (in mg) of the eliminated metabolites and the doses correlated with each other (r = 0.9334), whereas the percent ratio of 5-OH-flu and of 4'-OH-flu increased with dose. Half lives of renal elimination for flu, 5-OH-flu and 4'-OH-flu are largely independent of dose. The half life of flufenamic acid corresponds roughly to data from plasma levels (7-9 h), the two hydroxy derivatives are eliminated into urine with half lives from 15 to 24 h. The results show, that i.m. injection of an oily etofenamate solution follows a linear dose independent kinetic, while the amounts absorbed and renally eliminated are proportional to dose. The results correspond to plasma level studies.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1979

Effect of non-steroidal antiinflammatory drugs on some biological activities dependent on complement activation.

The paper presents the results of a study on the action of five non-steroidal antiinflammatory agents (phenylbutazone, indometacin, acetylsalicylic acid, niflumic and flufenamic acids) on two biological activities which are dependent on complement activation: opsonization of bacteria and membrane damage, the latter evaluated both with the classic immunohemolytic system and with a bactericidal assay. The three biological assays differ for complement sequences involved. Flufenamic and niflumic acids showed high inhibitory activity in the lytic assays. Human complement was more sensitive to inhibition than guinea-pig complement. Phagocytic test confirmed the inhibitory activity of flufenamic acid on complement dependent opsonization.

Animals↗

PubMed1986

Activated phase II metabolites: comparison of alkylation by 1-O-acyl glucuronides and acyl sulfates.

1-O-acyl glucuronides are reactive Phase II metabolites which can alkylate chemical nucleophiles. Industrial sulfate ester mixed anhydrides have been reported to be active acylating agents. This study was undertaken in order to establish that sulfate ester mixed anhydrides of clinically useful drugs could be synthesized, purified, and characterized as reactive chemical species. Their ability to alkylate 4-(p-nitrobenzyl)pyridine (NBP) and their stability in aqueous solution was compared with 1-O-acyl glucuronide conjugates of the same drugs. Synthesis of the 1-O-acyl glucuronides of the hypolipidemic agent, clofibric acid, and the nonsteroidal antiinflammatory drugs flufenamic acid and indomethacin were catalyzed by immobilized microsomal rabbit liver UDP-glucuronyltransferase. Potassium salts of the sulfate ester mixed anhydrides of these drugs were synthesized chemically by temperature-controlled reaction with chlorosulfonic acid in anhydrous pyridine. Half-lives at pH 2.0, 6.0, 7.4, and 10.0 were determined for each compound. The reactivity of the acyl glucuronides and sulfate ester mixed anhydrides towards the standard chemical nucleophile, 4-(p-nitrobenzyl pyridine (NBP), was measured using a spectrophotometric assay at several substrate concentrations. Acyl sulfate ester mixed anhydrides were shown to be 3-20 times more reactive towards NBP than their corresponding 1-O-acyl glucuronides. For both glucuronides and sulfate esters, relative reactivity towards NBP was: clofibric acid greater than indomethacin greater than flufenamic acid. This behavior paralleled the hydrolytic instability of the compounds.

Acylation↗

PubMed1997

Membrane depolarization in NRK fibroblasts by bradykinin is mediated by a calcium-dependent chloride conductance.

The effects of the phosphoinositide-mobilizing agonist bradykinin (BK) on membrane potential and intracellular calcium in monolayers of normal rat kidney (NRK) fibroblasts were investigated. BK induced a rapid transient depolarization in these cells, which was mimicked by other phosphoinositide-mobilizing factors such as prostaglandin F2alpha (PGF2alpha), lysophosphatidic acid (LPA), platelet-derived growth factor (PDGF-BB), and serum. Depolarization by BK was independent of extracellular Ca2+ or Na+. It was shown using extracellular Cl- substitutions that the depolarization was caused by an increased Cl- conductance. Depolarization was inhibited by 5-nitro-2-3-phenylpropyl(amino)benzoic acid (NPPB), niflumic acid, and flufenamic acid, inhibitors of calcium-dependent chloride channels. The depolarization provoked by BK could be mimicked by raising intracellular calcium with ionomycin or thapsigargin and could be blocked with geneticin, a blocker of phospholipase C. When intracellular calcium was buffered by loading the cells with 1,2-bis(2-aminophenoxy)ethane-NNN'N'-tetra-acetic acid (BAPTA), depolarization was prevented. We conclude that in NRK fibroblasts extracellular stimuli that increase intracellular calcium, depolarize the cells via the activation of a calcium-dependent chloride conductance. In addition to an increase in intracellular calcium, depolarization may be an important effector pathway in response to extracellular stimuli in fibroblasts. It is hypothesized that, in electrically coupled cells such as NRK fibroblasts, intercellular transmission of these depolarizations may represent a mechanism to coordinate uniform multicellular responses to Ca2+-mobilizing agonists.

Animals↗

PubMed1996

Chloride-channel block inhibits T lymphocyte activation and signalling.

Both large- and small-conductance chloride (Cl-) channels have been found in human T lymphocytes; however, apart from possible roles in mediating regulatory volume decrease, their functions are not understood. We have used patch-clamp electrophysiology, Ca2+ spectrofluorometry, and Western blot assay for phosphotyrosine to investigate the effects of blocking Cl- channels on proliferation and on specific events in the activation of normal human T cells. Four chemically distinct Cl- channel blockers inhibited both the small-conductance Cl- channels and phytohemagglutinin (PHA)-induced lymphocyte proliferation in a similar dose-dependent manner; their order of potency was 5-nitro-2(3-phenylpropylamino)-benzoic acid (NPPB) > 4,4'-diisothiocyano-2,2'-disulfonic acid (DIDS) > flufenamic acid >> IAA-94. The Cl- channel blockers inhibited both the PHA-induced mobilization of Ca2+ and the rapid tyrosine phosphorylation of several polypeptides. Cell proliferation was not rescued by the Ca+ ionophore ionomycin or by addition of exogenous interleukin-2 (IL-2). Moreover, the blockers also inhibited phosphotyrosine expression in IL-2-treated, activated lymphoblasts. Thus, our results support a role for Cl- channels in early, PHA-evoked signalling and in later, II-2-dependent stages of lymphocyte activation and proliferation.

Blotting, Western↗