Benzofurazan‐based fluorophore for the selective determination of flupentixol dihydrochloride: Application to content uniformity testing

One of the most commonly used drugs in treatment of schizophrenia is flupentixol dihydrochloride, therefore it is important to develop a simple, low cost and sensitive spectrofluorimetric method for the estimation of flupentixol dihydrochloride. The yellow fluorescent product that is generated from the nucleophilic substitution reaction of the free lone pair of the alcoholic hydroxyl group of the drug and 4‐chloro‐7‐nitrobenzofurazan (NBD‐Cl) in Mcllvaine buffer pH 7.0 was estimated at 510 nm (λ_ex 460 nm). The variables that affect the development of the reaction product were explored and optimized. The linear range of this method was 0.5–2.5 μg ml^?1 with a limit of quantitation equal to 0.29 μg ml^?1. Our method was successfully applied for the assurance of flupentixol in tablet form with average percentage recovery of 99.08 ± 1.01% without obstruction from the basic excipients exhibits. Furthermore, our strategy was extended to study the content uniformity testing of flupentixol in Fluaxnol^® tablets.     

Bumetanide-sensitive Ion Fluxes in Vascular Smooth Muscle Cells: Lack of Functional Na+, K+, 2 Cl− Cotransport

To examine the involvement of Na⁺,K⁺,2Cl⁻ cotransport in monovalent ion fluxes in vascular smooth muscle cells (VSMC), we compared the effect of bumetanide on ⁸⁶Rb, ³⁶Cl and ²²Na uptake by quiescent cultures of VSMC from rat aorta. Under basal conditions, the values of bumetanide-sensitive (BS) inward and outward ⁸⁶Rb fluxes were not different. Bumetanide decreased basal ⁸⁶Rb uptake by 70–75% with a K _i of ∼0.2–0.3 μm. At concentrations ranging up to 1 μm, bumetanide did not affect ³⁶Cl influx and reduced it by 20–30% in the range from 3 to 100 μm. In contrast to ⁸⁶Rb and ³⁶Cl influx, bumetanide did not inhibit ²²Na uptake by VSMC. BS ⁸⁶Rb uptake was completely abolished in Na⁺- or Cl⁻-free media. In contrast to ⁸⁶Rb, basal BS ³⁶Cl influx was not affected by Na⁺ _o and K⁺ _o . Hyperosmotic and isosmotic shrinkage of VSMC increased ⁸⁶Rb and ³⁶Cl influx to the same extent. Shrinkage-induced increments of ⁸⁶Rb and ³⁶Cl uptake were completely abolished by bumetanide with a K _i or ∼0.3 μm. Shrinkage did not induce BS ⁸⁶Rb and ³⁶Cl influx in (Na⁺ or Cl⁻)- and (Na⁺ or K⁺)-depleted media, respectively. In the presence of an inhibitor of Na⁺/H⁺ exchange (EIPA), neither hyperosmotic nor isosmotic shrinkage activated ²²Na influx. Bumetanide (1 μm) did not modify basal VSMC volume and intracellular content of sodium, potassium and chloride but abolished the regulatory volume increase in isosmotically-shrunken VSMC. These data demonstrate the absence of the functional Na⁺,K⁺,2Cl⁻ cotransporter in VSMC and suggest that in these cells basal and shrinkage-induced BS K⁺ influx is mediated by (Na⁺ _o + Cl⁻ _o )-dependent K⁺/K⁺ exchange and Na⁺ _o -dependent K⁺,Cl⁻ cotransport, respectively. Received: 30 January 1996/Revised: 20 May 1996     

A Cl channel in Ascaris suum selectivity conducts dicarboxylic anion products of glucose fermentation and suggests a role in removal of waste organic anions

The permeability of organic anions (produced from anaerobic fermentation of glucose) through a nonselective membrane Cl channel was examined. Single channel recording techniques were used to study the permeabilities of the anions: oxalate, succinate, oxaloacetate, malate, lactate and pyruvate in Ascaris muscle cell membranes. All of the anions, except malate, were found to be conducted through the channel. The relative permeability of most anions could be predicted from the component structure of the anions. The failure of the channel to conduct malate prevents an energy drain on the cell. These studies further the hypothesis that a Cl channel functions to transport waste organic anions across the cell membrane. This mechanism does not require specific exchange carriers for the anions. Channels with properties like the nonselective anion channels of Ascaris, are suitable for transport of carboxylic anions through cell membranes, down a concentration or potential gradient.This work was financed by the Scientific and Engineering Research Council (S.E.R.C.).     

The chloride concentration in the lateral intercellular spaces of MDCK cell monolayers

We measured the Cl concentration of the lateral intercellular spaces (LIS) of MDCK cell monolayers, grown on glass coverslips, by video fluorescence microscopy. Monolayers were perfused at 37°C either with HEPES-buffered solutions containing 137 mm Cl or bicarbonate/CO₂-buffered solutions containing 127 mm Cl. A mixture of two fluorescent dyes conjugated to dextrans (MW 10,000) was microinjected into domes and allowed to diffuse into the nearby LIS. The Cl sensitive dye, ABQ-dextran, was selected because of its responsiveness at high Cl concentrations; a Clinsensitive dye, Cl-NERF-dextran, was used as a reference. Both dyes were excited at 325 nm, and ratios of the fluorescence intensity at spectrally distinct emission wavelengths were obtained from two intensified CCD cameras, one for ABQ-dextran the other for Cl-NERFdextran. LIS Cl concentration was calibrated in situ by treating the monolayer with digitonin or ouabain and varying the perfusate Cl between 0 and 137 mm (HEPES buffer) or between 0 and 127 mm (bicarbonate/CO₂ buffer). LIS Cl in HEPES-buffered solutions averaged 176 ± 19 mm (n = 12), calibrated with digitonin, and 170 ± 9 mm (n = 12), calibrated with ouabain. LIS Cl in bicarbonate/CO₂-buffered solutions averaged 174 ± 10 mm (n = 7) using the ouabain calibration. The Cl concentration of MDCK cell domes, measured with Clsensitive microelectrodes and by microspectrofluorimetry, did not differ significantly. Images of the LIS at 3 focal planes, near the tight junction, midway and basal, failed to reveal any gradients in Cl concentration along the LIS. LIS Cl changed rapidly in response to perfusate Cl with characteristic times of 0.8 ± 0.1 min (n = 21) for Cl decrease and 0.3 ± 0.04 min (n=21) for Cl increase. In conclusion, (i) Cl concentration is higher in the LIS than in the bathing medium, (ii) no gradients of Cl along the depth of LIS are detectable, (iii) junctional Cl permeability is high.We gratefully acknowledge the assistance of Mr. Richard D'Alessandro in the performance of the microelectrode studies. Mr. Carter Gibson designed the electronics and wrote the key computer programs used in this study. The authors are grateful to Dr. Alan Verkman (UCSF) for his advice and gifts of fluorescent probes in the early stages of this work.     

Effect of protease inhibitors on protein degradation in rat hepatoma cells II. Effects on ornithine decarboxylase and tyrosine aminotransferase

Tosyllysine chloromethyl ketone and tosylphenylalanine chloromethyl ketone in vitro are active-site specific and irreversible inhibitors of trypsin (EC 3.4.21.4) and chymotrypsin (EC. 3.4.21.1) respectively. Using rat hepatoma cells in suspension culture, both inhibitors were found to partially inhibit breakdown of prelabelled cell proteins ot amino acids, the effect being greastest in the absence of serum. Protein synthesis in rat hepatoma cells, reticulocytes and reticulyte lysates was also irreversibly inhibited by these compounds. Reduction of ATP levels with antimycin a inhibited protein degradation, but neither tosylphenylalanine chloromethyl ketone nor tosyllysine chloromethyl ketone had any effect on ATP concentration in rat hepatoma cells. These results suggest that the degradation of at least some proteins in animal cells may involve the action of serine protease(s).     

Effect of membrane potential on furosemide-inhibitable sodium influxes in human red blood cells

Summary Furosemide-inhibitable Na influx (a measure of Na/K/Cl cotransport) was determined as a function of membrane potential in human red blood cells. The membrane potential was varied from –42 to +118 mV using valinomycin and gradients of K. The furosemide-inhibitable, unidirectional Na influx was independent of membrane potential over the entire range of potentials. The change in flux per mV, 0.443 mol/(liter cells·hr· mV), was not significantly different from zero. The mean flux was 153±16mol/(liter cells·hr) (±sem,n=71). The ouabain and furosemide-resistant influexes of Na and K were also measured as functions of membrane potential using either valinomycin and K or a chloride-free, tartrate flux medium to vary membrane potential. The unidirectional Na influx decreased slightly as the membrane potential was increased from negative potentials to about +10 mV. At higher membrane potentials Na influx rose dramatically with potential. This increase was not reversible and was also observed with K influx.     

Modes of Cl− transport across the mucosal and serosal membranes of urodele intestinal cells

Summary The characteristics of Cl^– movement across luminal and basolateral membranes ofAmphiuma intestinal absorptive cells were studied using Cl^–-sensitive microelectrodes and tracer³⁶Cl techniques. Intracellular Cl^– activity (a _Cl ⁱ ) was unchanged when serosal Cl^– was replaced; when luminal Cl^– was replaced cell Cl^– was rapidly lost. Accordingly, the steady statea _Cl ⁱ could be varied by changing the luminal [Cl]. As luminal [Cl] was raised from 1 to 86mM,a _Cl ⁱ rose in a linear manner, the mucosal membrane hyperpolarized, and the transepithelial voltage became serosa negative. In contrast, the rate of Cl^– transport from the cell into the serosal medium, measured as the SITS-inhibitable portion of the Cl^– absorptive flux, attained a maximum whena _Cl ⁱ reached an apparent value of 17mm, indicating the presence of a saturable, serosal transport step. The stilbeneinsensitive absorptive flux was linear with luminal [Cl], suggestive of a paracellular route of movement. Intracellulara _Cl was near electrochemical equilibrium at all but the lowest values of luminal [Cl] after interference produced by other anions was taken into account.a _Cl ⁱ was unaffected by Na replacement, removal of medium K, or elevation of medium HCO ₃ ^– . Mucosae labeled with³⁶Cl lost isotope into both luminal and serosal media at the same rate and from compartments of equal capacity. Lowering luminal [Cl] or addition of theophylline enhanced luminal Cl^– efflux. It is concluded that a conductive Cl leak pathway is present in the luminal membrane. Serosal transfer is by a saturable, stilbene-inhibitable pathway. Luminal Cl^– entry appears to be passive, but an electrogenic uptake cannot be discounted.     

Actions of avermectin B1a on the γ-aminobutyric AcidA receptor and chloride channels in rat brain

The interaction of avermectin B_1a (AVM) with the γ-aminobutyric acid (GABA) receptor of rat brain was studied using radioactive ligand binding and tracer ion flux assays. Avermectin potentiated the binding of [³H]flunitrazepam and inhibited the binding of both [³H]muscimol and [³⁵S]t-butylbicyclo-phosphorothionate to the GABA_A receptor. Inhibition of muscimol binding by AVM suggested competitive displacement. Two kinds of ³⁶chloride (Cl) flux were studied. The ³⁶Cl efflux from preloaded microsacs was potentiated by AVM and was highly inhibited by the Cl-channel blocker 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS). However, it was not potentiated by GABA nor was it sensitive to the convulsants picrotoxin or bicuculline. On the other hand, ³⁶Cl-influx measurement in a different microsac preparation of rat brain was very sensitive to GABA and other GABA-ergic drugs. Avermectin induced ³⁶Cl influx into these microsacs in a dose–dependent manner, but to only 35% of the maximal influx induced by GABA. The AVM-induced ³⁶Cl influx was totally blocked by bicuculline. It is suggested that AVM opens the GABA_A-receptor Cl channel by binding to the GABA recognition site and acting as a partial receptor agonist, and also opens a voltage–dependent Cl channel which is totally insensitive to GABA but is very sensitive to DIDS.     

Activation of chloride conductance in pig jejunal brush border vesicles

Summary Requirements for the activation of Cl conductance have been investigated in pig jejunal brush border vesicles. The stability of ATP as a substrate for protein kinase activity, the stability of the phosphoprotein product of protein kinase action, and the choice of buffer system used for vesicle preparation were studied as variables which affected the outcome of in vitro activation attempts. Arsenate was selected as the most effective agent in protecting ATP from hydrolysis by the phosphatase activity in this vesicle system. Brush border vesicle protein appeared to prevent the accumulation of phosphoprotein in a cAMP-dependent protein kinase reaction, and vesicle protein only had phosphate acceptor activity when KF was added as a presumptive inhibitor of phosphoprotein phosphatase.A Cl conductance response to a potassium gradient and valinomycin was present in vesicles prepared in buffers containing tetramethylammonium. Cl^– conductance activity was not increased in this system by the addition of ATP, dibutyryl cyclic AMP, and cyclic AMP-dependent protein kinase.There was no Cl conductance response to a potassium gradient in vesicles buffered with imidazolium-acetate. Incorporation of ATP, AsO ₄ ^3– , and F^– into these nonconductive vesicles by homogenization, followed by addition of dibutyryl cAMP, produced substantial conductance activity. Maximal activation of Cl^– conductance was obtained with vesicles prepared in imidazolium-acetate buffering, using precautions to stabilize ATP and phosphoprotein prior to conductance measurements.     

Effect of changes of pHi on intracellular calcium in a smooth muscle-like cell line

The effect of changes of pH_i on Ca_i were studied using fluorescent dyes in cells of the cultured smooth muscle-like line, BC3H-1. Resting Ca_i in these cells was 182 ± 12 nM (n = 74) at pH_o of 7.4. Upon exposure to NH₄Cl, which rapidly alkalinized cells, a transient increase of Cai to 349 ± 55 nM (n = 29) was observed. The peak of the transient occurred within 30 s of exposure to NH₄Cl and returned to baseline within 1 minute. Two other procedures which resulted in rapid cellular alkalinization also caused a transient rise in Ca_i: exposure to and then removal of CO₂ (Ca_i increased from 182 ± 22 to 248 ± 28 nM; n = 8); and exposure to and then removal of Na propionate (Ca_i increased from 242 ± 32 to 456 ± 71 nM; n = 9). The NH4Cl-induced Ca_i transient was eliminated by exposure to 0.2 mM TMB8 and to Ca-free solutions, but not by exposure to 0.5 mM LaCl₃. Sustained changes of pH_i can be induced by varying pH_o. When pH_o was lowered to 6.9, Ca_i fell by 49 ± 11 nM but increased by 203 ± 51 nM (n = 6) when pH_o was raised to 7.9. These data indicate that rapid alkalinization of BC3H-1 cells results in a rapid transient rise of Ca_i. This transient is most likely due to the release of Ca from intracellular stores but may also involve an increase of Ca influx. Steady state values of Cai are positively correlated with steady state pH_i. These data may have implications for the contractile state of smooth muscle during periods of acid/base disturbances and relate to the role of elevated pH_i in cells from hypertensive animals.