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PubMed2002

Chloride transport in rabbit esophageal epithelial cells.

We investigated Cl(-) transport pathways in the apical and basolateral membranes of rabbit esophageal epithelial cells (EEC) using conventional and ion-selective microelectrodes. Intact sections of esophageal epithelium were mounted serosal or luminal side up in a modified Ussing chamber, where transepithelial potential difference and transepithelial resistance could be determined. Microelectrodes were used to measure intracellular Cl(-) activity (a), basolateral or apical membrane potentials (V(mBL) or V(mC)), and the voltage divider ratio. When a basal cell was impaled, V(mBL) was -73 +/- 4.3 mV and a(i)(Cl) was 16.4 +/- 2.1 mM, which were similar in presence or absence of bicarbonate. Removal of serosal Cl(-) caused a transient depolarization of V(mBL) and a decrease in a(i)(Cl) of 6.5 +/- 0.9 mM. The depolarization and the rate of decrease of a(i)(Cl) were inhibited by approximately 60% in the presence of the Cl(-)-channel blocker flufenamate. Serosal bumetanide significantly decreased the rate of change of a(i)(Cl) on removal and readdition of serosal Cl(-). When a luminal cell was impaled, V(mC) was -65 +/- 3.6 mV and a was 16.3 +/- 2.2 mM. Removal of luminal Cl(-) depolarized V(mC) and decreased a by only 2.5 +/- 0.9 mM. Subsequent removal of Cl(-) from the serosal bath decreased a(i)(Cl) in the luminal cell by an additional 6.4 +/- 1.0 mM. A plot of V(mBL) measurements vs. log a(i)(Cl)/log a(o)(Cl) (a(o)(Cl) is the activity of Cl(-) in a luminal or serosal bath) yielded a straight line [slope (S) = 67.8 mV/decade of change in a(i)(Cl)/a(o)(Cl)]. In contrast, V(mC) correlated very poorly with log a/a (S = 18.9 mV/decade of change in a/a). These results indicate that 1) in rabbit EEC, a(i)(Cl) is higher than equilibrium across apical and basolateral membranes, and this process is independent of bicarbonate; 2) the basolateral cell membrane possesses a conductive Cl(-) pathway sensitive to flufenamate; and 3) the apical membrane has limited permeability to Cl(-), which is consistent with the limited capacity for transepithelial Cl(-) transport. Transport of Cl(-) at the basolateral membrane is likely the dominant pathway for regulation of intracellular Cl(-).

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2003

Fundamental role of ClC-3 in volume-sensitive Cl- channel function and cell volume regulation in AGS cells.

Volume regulation is essential for cell function, but it is unknown which channels are involved in a regulatory volume decrease (RVD) in human gastric epithelial cells. Exposure to a hypotonic solution caused the increase in AGS cell volume, followed by the activation of a current. The reversal potential of the swelling-induced current suggested that Cl- was the primary charge carrier. The selectivity sequence for different anions was I- > Br- > Cl- > F- > gluconate. This current was inhibited by flufenamate, DIDS, tamoxifen, and 5-nitro-2-(3-phenylpropylamino)benzoate. Intracellular dialysis of three different anti-ClC-3 antibodies abolished or attenuated the Cl- current and disrupted RVD, whereas the current and RVD was unaltered by anti-ClC-2 antibody. Immunoblot studies demonstrated the presence of ClC-3 protein in Hela and AGS cells. RT-PCR analysis detected expression of ClC-3, MDR-1, and pICln mRNA in AGS cells. These results suggest a fundamental role of endogenous ClC-3 in the swelling-activated Cl- channels function and cell volume regulation in human gastric epithelial cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

Assay characteristics and fibrin affinity of plasminogen activators of the intrinsic fibrinolytic system.

Plasminogen activators (PA) in the euglobulin fraction of dextran sulfate activated plasma (DS-EF) were assayed on fibrin plates. Activity related to tissue plasminogen activator (t-PA) or urokinase (u-PA) was quantified by antiserum inhibition. The DS-EF contained 30% t-PA, 30% u-PA and 40-50% activity unrelated to t-PA or u-PA. The latter was completely inhibited by 1.7 mumol/1 C1-inhibitor (C1INH), the two former were less sensitive. Addition of flufenamate to the DS-EF (DS-EF/Fluf) from normal and two factor XII (F XII)-deficient plasmas increased their activities to the same high level. More than 50% of the activity was unrelated to t-PA or u-PA, 30-40% was u-PA and 5-10% t-PA related. After addition of fibrinogen to DS-EF/Fluf and clotting with thrombin, the remaining solution contained only about 30% of the total activity, including less than 10% u-PA. The epsilon-aminocaproic acid inhibition pattern obtained with the DS-EF was uniform, and thus different from the biphasic pattern obtained with the low fibrin affinity PA, two-chain urokinase. Thus, both the plasma u-PA and the major unidentified PA in plasma have affinity for fibrin.

Adult↗
PubMed1988

Properties of the principal anion-exchange mechanism in human neutrophils.

As part of our ongoing studies aimed at understanding the physiology of human neutrophil function, we have been investigating the nature of the principal anion-exchange mechanism in these cells. This countertransport system functions physiologically as a Cl-/HCO3- exchanger in intracellular pH regulation from alkalinization (Simchowitz and Roos, 1985). In summarizing the results of our work, it is worthwhile to keep in mind the well-defined properties of the classic inorganic anion-exchange carrier of red blood cells, as important differences between the two are evident. Inhibitor Profile In our original articles on Cl- movements (Simchowitz and De Weer, 1986; Simchowitz et al., 1986), we had identified CHC, which suppresses monocarboxylate transport in mitochondria (Halestrap and Denton, 1975), as a relatively weak inhibitor. Several structural analogues, including alpha-methylcinnamate and alpha-phenylcinnamate, exhibited enhanced activity against anion exchange. The most potent compound thus far identified was UK-5099, which was approximately 300-fold more active than CHC. The inhibitory effect of each member of this series of compounds was strictly competitive in nature: both the drug and Cl ions appeared to bind to the same external translocation site on the exchange carrier. The inhibition by several other unrelated agents (e.g., MK-473, niflumate, flufenamate, and NAP-taurine), all weak organic acids, was also of the competitive type, which suggests that these compounds bind to the carrier by virtue of their negatively charged carboxyl or sulfonic groups. In marked contrast to erythrocytes, the anion-exchange carrier of neutrophils is rather insensitive to the disulfonic stilbenes SITS and DIDS (Simchowitz and De Weer, 1986): little or no effect can be detected in 148 mM Cl-. However, under conditions of low external Cl- (5-35 mM), SITS can in fact be shown to interact weakly with the anion binding site of the exchange carrier. In a comparable vein, several other drugs, all of which have been reported to suppress Cl- fluxes in red cells (for reviews, see Sachs et al., 1975; Gunn, 1979; Knauf, 1979; Lowe and Lambert, 1983), were either completely inactive against anion exchange in neutrophils at a concentration of 1 mM or only marginally effective at doses several orders of magnitude greater than those required in red blood cells. This list included furosemide, picrylsulfonate, maleic anhydride, 5,5'-dithio-bis(2-nitrobenzoate), salicylate, 2-methoxy-5-nitrotropone, and aldrithiol.4+he external

Anion Exchange Resins↗
PubMed1998

Basal chloride currents in murine airway epithelial cells: modulation by CFTR.

We have isolated ciliated respiratory cells from the nasal epithelium of wild-type and cystic fibrosis (CF) null mice and used the patch-clamp technique to investigate their basal conductances. Current-clamp experiments on unstimulated cells indicated the presence of K+ and Cl- conductances and, under certain conditions, a small Na+ conductance. Voltage-clamp experiments revealed three distinct Cl- conductances. Itv-indep was time and voltage independent with a linear current-voltage (I-V) plot; Iv-act exhibited activation at potentials greater than +/- 50 mV, giving an S-shaped I-V plot; and Ihyp-act was activated by hyperpolarizing potentials and had an inwardly rectified I-V plot. The current density sequence was Ihyp-act = Iv-act >> Itv-indep. These conductances had Cl(-)-to-N-methyl-D-glucamine cation permeability ratios of between 2.8 and 10.3 and were unaffected by tamoxifen, flufenamate, glibenclamide, DIDS, and 5-nitro-2-(3-phenylpropylamino) benzoic acid but were inhibited by Zn2+ and Gd3+. Itv-indep and Iv-act were present in wild-type and CF cells at equal density and frequency. However, Ihyp-act was detected in only 3% of CF cells compared with 26% of wild-type cells, suggesting that this conductance may be modulated by cystic fibrosis transmembrane conductance regulator (CFTR).

Animals↗
PubMed1998

The substrate recognition domain in the Na+/dicarboxylate and Na+/sulfate cotransporters is located in the carboxy-terminal portion of the protein.

The Na+/dicarboxylate cotransporter, NaDC-1, and the Na+/sulfate cotransporter, NaSi-1, share 43% sequence identity, but they exhibit no overlap in substrate specificity. A functional chimera, SiDC-4, was prepared from NaDC-1 and NaSi-1 by homologous recombination and expressed in Xenopus oocytes. SiDC-4 contains putative transmembrane domains 1-4 of NaSi-1 (amino acids 1-139) and putative transmembrane domains 5-11 of NaDC-1 (amino acids 141-593). SiDC-4 retains the substrate specificity of NaDC-1, which suggests that the substrate recognition domain is found in the carboxy-terminal portion of the protein, past amino acid 141. However, residues that affect substrate affinity and inhibition by furosemide and flufenamate are found in the amino terminal third of the protein. The cation binding properties of SiDC-4, including a stimulation of transport by lithium, differed from both parental transporters, suggesting that cation binding is determined by interactions between the amino- and carboxy-terminal portions of the protein. We conclude that the substrate recognition site of NaDC-1 and NaSi-1 is found in the carboxy-terminal portion of the protein, past amino acid 141, but residues in the amino terminus can affect substrate affinity, inhibitor sensitivity, and cation selectivity.

Animals↗
PubMed2002

Sodium fluxes through nonselective cation channels in the plasma membrane of protoplasts from Arabidopsis roots.

The aim of the present work was to characterize Na(+) currents through nonselective cation channels (NSCCs) in protoplasts derived from root cells of Arabidopsis. The procedure of the protoplast isolation was modified to increase the stability of Arabidopsis root protoplasts in low external Ca(2+) by digesting tissue in elevated Ca(2+). Experiments in whole-cell and outside-out modes were carried out. We found that Na(+) currents in Arabidopsis root protoplasts were mediated by cation channels that were insensitive to externally applied tetraethylammonium(+) and verapamil, had no time-dependent activation (permanently opened or completely activated within 1-2 ms), were voltage independent, and were weakly selective for monovalent cations. The selectivity sequence was as follows: K(+) (1.49) > NH(4)(+) (1.24) > Rb(+) (1.15) approximately equal to Cs(+) (1.10) approximately equal to Na(+) (1.00) > Li(+) (0.73) > tetraethylammonium(+) (0.47). Arabidopsis root NSCCs were blocked by H(+) (pK approximately equal to 6.0), Ca(2+) (K(1/2) approximately equal to 0.1 mM), Ba(2+), Zn(2+), La(3+), Gd(3+), quinine, and the His modifier diethylpyrocarbonate. They were insensitive to most organic blockers (nifedipine, verapamil, flufenamate, and amiloride) and to the SH-group modifier p-chloromercuriphenyl sulfonic acid. Voltage-insensitive, Ca(2+)-sensitive single channels were also resolved. Properties of Arabidopsis root NSCCs are discussed and compared with characteristics of similar conductances studied previously in plants and animals. It is suggested that NSCCs present a distinct group of plant ion channels, mediating toxic Na(+) influx to the cell and probably having other important roles in physiological processes of plants.

Arabidopsis↗
PubMed1995

Ca2+ influx induced by store release and cytosolic Ca2+ chelation in Ht29 colonic carcinoma cells.

Cl- secretion in HT29 cells is regulated by agonists such as carbachol, neurotensin and adenosine 5'-triphosphate (ATP). These agonists induce Ca2+ store release as well as Ca2+ influx from the extracellular space. The increase in cytosolic Ca2+ enhances the Cl- and K+ conductances of these cells. Removal of extracellular Ca2+ strongly attenuates the secretory response to the above-mentioned agonists. The present study utilises patch-clamp methods to characterise the Ca2+ influx pathway. Inhibitors which have been shown previously to inhibit non-selective cation channels, such as flufenamate (0.1 mmol.l-1, n = 6) and Gd3+ (10 micromol.l-1, n=6) inhibited ATP (0.1 mmol.l-1) induced increases in whole-cell conductance (Gm). When Cl- and K+ currents were inhibited by the presence of Cs2SO4 in the patch pipette and gluconate in the bath, ATP (0.1 mmol.l-1) still induced a significant increase in Gm from 1.2 +/- 0.3 nS to 4.7 +/- 1 nS (n = 24). This suggests that ATP induces a cation influx with a conductance of approximately 3-4 nS. This cation influx was inhibited by flufenamate (0.1 mmol.l-1, n = 6) and Gd3+ (10 micromol.l-1, n = 9). When Ba2+ (5 mmol.l-1) and 4,4'-diisothiocyanato-stilbene-2-2'-disulphonic acid (DIDS, 0.1 mmol.l-1) were added to the KCl/K-gluconate pipette solution to inhibit K+ and Cl- currents and the cells were clamped to depolarised voltages, ATP (0.1 mmol.l-1) reduced the membrane current (Im) significantly from 86 +/- 14 pA to 54 +/- 11 pA (n = 13), unmasking a cation inward current. In another series, the cation inward current was activated by dialysing the cell with a KCl/K-gluconate solution containing 5-10 mmol.l-1 1,2-bis-(2-aminoethoxy)ethane-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis-(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA). The zero-current membrane voltage (Vm) and Im (at a clamp voltage of +10 mV) were monitored as a function of time. A new steady-state was reached 30-120 s after membrane rupture. Vm depolarised significantly from -33 +/- 2 mV to -12 +/- 1 mV, and Im fell significantly from 17 +/- 2 pA to 8.9 +/- 1.0 pA (n = 71). This negative current, representing a cation inward current, was activated when Ca2+ stores were emptied and was reduced significantly ( Im) when Ca2+ and/or Na+ were removed from the bathing solution: removal of Ca2+ in the absence of Na+ caused a Im of 5.0 +/- 1.2 pA (n = 12); removal of Na+ in the absence of Ca2+ caused a Im of 12.8 +/- 3.5 pA (n = 4). The cation inward current was also reduced significantly by La3+, Gd3+, and flufenamate. We conclude that store depletion induces a Ca2+/Na+ influx current in these cells. With 145 mmol.l-1 Na+ and 1 mmol.l-1 Ca2+, both ions contribute to this cation inward current. This current is an important component in the agonist-regulated secretory response.

Adenosine Triphosphate↗
PubMed2003

20-hydroxyeicosatetraenoic acid (20-HETE) activates mouse TRPC6 channels expressed in HEK293 cells.

In the present study, we show that the eicosanoid compound, 20-hydroxyeicosatetraenoic acid (20-HETE), an important arachidonic acid metabolite, activates mouse TRPC6 in a stable, overexpressing HEK293 cell line, Hek-t6.11. Application of 20-HETE rapidly induced an inward, non-selective current in whole-cell recordings, which was inhibited by N-methyl-d-glucamine, 1.8 mm Ca2+, and 100 microM Gd3+ but remained unaffected by flufenamate and indomethacin. The current-voltage relationship obtained at low concentrations of 20-HETE (1-10 microM) demonstrated slight inward rectification, whereas the highest concentration of 20-HETE tested (30 microM) showed outward rectification, as shown previously for these channels using 100 microM 1-oleoyl-2-acetyl-sn-glycerol. Dose-response curves indicate that 20-HETE activated TRPC6 channels with an EC50 = 0.8 microM. Single channel analysis using inside-out patches revealed that 20-HETE increased open probability of mouse TRPC6 channels approximately 3-fold, and this was in a membrane-delimited fashion. Interestingly, 20-HETE did not provoke changes in intracellular Ca2+ concentrations. Thus, we have identified an arachidonic acid metabolite, 20-HETE, as a novel activator for a TRP family member, TRPC6.

Animals↗
PubMed1996

Volume-sensitive taurine efflux from mammary tissue is not obliged to utilize volume-activated anion channels.

Cell-swelling, induced by a hyposmotic shock, activates the release of taurine from lactating rat mammary tissue explants. The degree of stimulation of taurine efflux was dependent upon the extent of cell-swelling. Volume-sensitive taurine release was attenuated by the anion transport inhibitors NPPB, DIOA, DIDS, niflumate, flufenamate, mefenamate and diiodosalicylate but not by salicylate. Cell-swelling, following a hyposmotic challenge, did not increase the unidirectional efflux of radiolabelled I- or D-asparate from mammary tissue explants. The results suggest that although mammary tissue expresses a volume-sensitive amino acid transport system which is inhibited by anion transport blockers the pathway has no identity with volume-activated anion channels.

Animals↗
PubMed2000

A calcium-activated cation current by an alternatively spliced form of Trp3 in the heart.

To investigate a cDNA encoding cation current, we isolated an alternatively spliced form of a rat Trp3, designated Trp3sv. Trp3sv encodes 736 amino acids with a unique N terminus and six transmembrane segments. Expression of the cRNA in Xenopus oocytes was successfully performed. The cation selective current appeared after the addition of ionomycin or induced by prolonged depolarization but not by hyperpolarization. This induction was not observed by a treatment with thapsigargin, phorbol ester, or ATP. Na(+), K(+), tetraethylammonium, and divalent cations were permeable, while N-methylglucamine and chloride were nominally impermeable ions. The currents were not inhibited by flufenamate ruthenium red but nonspecifically by 2 mm Gd(3+). Northern as well as Western blot suggested lower levels of the expression observed in some organs, while reverse transcriptase-polymerase chain reaction suggested that it widely spread among various organs. Therefore, we may conclude that N-terminal spliced valiant of Trp3, Trp3sv, encodes a calcium-activated cation channel in various organs.

Adenosine Triphosphate↗
PubMed1989

The involvement of lactate and calcium as mediators of the electrical and mechanical responses of the myocardium to conditions of simulated ischaemia.

1. Rat isolated and superfused atria were exposed to a lactate-containing solution simulating the composition of extracellular fluid during myocardial ischaemia (SI). 2. Atria subjected to SI showed a decreased adenosine 5'-triphosphate (ATP) content, a rise in diastolic tension, a diminished conduction velocity of action potentials and shortened refractory periods. All these changes were less pronounced during lactate-free SI. 3. Atria preloaded with calcium displayed exaggerated responses measured electrically and mechanically during exposure to SI, whereas atria previously depleted of calcium displayed diminished electrical and mechanical responses to SI. Neither calcium loading nor calcium depletion modified the SI-induced depletion of the atrial stores of ATP. 4. Sulphinpyrazone protected atria against all aspects of the response to SI, but failed to protect the muscle under conditions of lactate-free SI. It is concluded that during SI, sulphinpyrazone protects against a lactate-mediated inhibition of the glycolytic synthesis of ATP. 5. Flufenamate exaggerated all responses of the atria to SI. These deleterious actions were still observed during lactate-free SI. It is concluded that flufenamate inhibits the synthesis of ATP in the mitochondria.

Adenosine Triphosphate↗
PubMed1989

Topography of prostaglandin H synthase. Antiinflammatory agents and the protease-sensitive arginine 253 region.

Prostaglandin H synthase catalyzes two reactions: the bis-dioxygenation of arachidonic acid to form prostaglandin G2 (cyclooxygenase activity), and the reduction of hydroperoxides to the corresponding alcohols (peroxidase activity). The cyclooxygenase activity can be selectively inhibited by many nonsteroidal antiinflammatory agents including indomethacin. In the native synthase, there is a single prominent protease-sensitive region, located near Arg253; binding of the heme prosthetic group makes the synthase resistant to proteases. To investigate the spatial relationship between the area of the synthase which interacts with indomethacin and the protease-sensitive region, the effects of indomethacin and similar agents on the protease sensitivity of the two enzymatic activities and of the synthase polypeptide were examined. Incubation of the synthase apoenzyme with trypsin (3.6% w/w) resulted in the time-dependent coordinate loss (75% at 1 h) of both enzymatic activities and the cleavage (85% at 1 h) of the 70-kDa subunit into 38- and 33-kDa fragments, indicating that proteolytic cleavage of the polypeptide at Arg253, destroyed both activities of the synthase simultaneously. Indomethacin, (S)-flurbiprofen, or meclofenamate (each at 20 microM) rendered both activities and the synthase polypeptide (at 5 microM subunit) resistant to attack by trypsin or proteinase K; these agents also inhibited the cyclooxygenase activity of the intact synthase. Two reversible cyclooxygenase inhibitors, ibuprofen and flufenamate, also made both of the activities and the synthase polypeptide more resistant to trypsin. Titration of the apoenzyme with indomethacin (0-3 mol/mol of synthase dimer) resulted in proportional increases in the inhibition of the cyclooxygenase and in the resistance to attack by trypsin. (R)-Flurbiprofen did not increase the resistance to protease or appreciably inhibit the cyclooxygenase. These results suggest that the same stereospecific interaction of these agents with the synthase that produced inhibition of the cyclooxygenase led to a decreased accessibility of the Arg253 region to proteases. Aspirin treatment made the synthase less resistant to trypsin; aspirin-treated synthase became more resistant to trypsin when it was incubated with indomethacin before addition of the protease. The presence of 50 microM arachidonate during digestion of apoenzyme or aspirin-treated apoenzyme with trypsin did not decrease the cleavage of the synthase subunit.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylation↗
PubMed1986

A new method for the reconstitution of the anion transport system of the human erythrocyte membrane.

The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent Triton X-100. It was incorporated into spherical lipid bilayers by the following procedure: Dry phosphatidylcholine was suspended in the protein solution. Octylglucopyranoside was added until the milky suspension became clear. The sample was dialyzed overnight against detergent-free buffer. Residual Triton X-100 was removed from the opalescent vesicle suspension by sucrose density gradient centrifugation and subsequent dialysis. Sulfate efflux from the vesicles was studied, under exchange conditions, using a filtration method. Three vesicle subpopulations could be distinguished by analyzing the time course of the efflux. One was nearly impermeable to sulfate, and efflux from another was due to leaks. The largest subpopulation, however, showed transport characteristics very similar to those of the anion transport system of the intact erythrocyte membrane: transport numbers (at 30 degrees C) close to 20 sulfate molecules per band 3 and min, an activation energy of approx. 140 kJ/mol, a pH maximum at pH 6.2, saturation of the sulfate flux at sulfate concentrations around 100 mM, inhibition of the flux by H2DIDS and flufenamate (approx. KI-values at 30 degrees C: 0.1 and 0.7 microM, respectively), and "right-side-out" orientation of the transport protein (as judged from the inhibition of sulfate efflux by up to 98% by externally added H2DIDS). Thus, the system represents, for the first time, a reconstitution of all the major properties of the sulfate transport across the erythrocyte membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1999

Ionic basis for plateau potentials in deep dorsal horn neurons of the rat spinal cord.

Approximately 28% of dorsal horn neurons (DHNs) in lamina V of the rat spinal cord generate voltage-dependent plateau potentials underlying accelerating discharges and prolonged afterdischarges in response to steady current pulses or stimulation of nociceptive primary afferent fibers. Using intracellular recordings in a transverse slice preparation of the cervical spinal cord, we have analyzed the ionic mechanisms involved in the generation and maintenance of plateau potentials in lamina V DHNs. Both the accelerating discharges and afterdischarges were reversibly blocked by Mn(2+) and enhanced when Ca(2+) was substituted with Ba(2+). The underlying tetrodotoxin-resistant regenerative depolarization was sensitive to dihydropyridines, being blocked by nifedipine and enhanced by Bay K 8644. Substitution of extracellular Na(+) with N-methyl-D-glucamine or choline strongly decreased the duration of the plateau potential. Loading the neurons with the calcium chelator BAPTA did not change the initial response but clearly decreased the maximum firing frequency and the duration of the afterdischarge. A similar effect was obtained with flufenamate, a specific blocker of the calcium-activated nonspecific cation current (I(CAN)). We conclude that the plateau potential of deep DHNs is supported by both Ca(2+) influx through intermediate-threshold voltage-gated calcium channels of the L-type and by subsequent activation of a CAN current. Ca(2+) influx during the plateau is potentially of importance for pain integration and the associated sensitization in spinal cord.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

Expression of a glutamate-activated chloride current in Xenopus oocytes injected with Caenorhabditis elegans RNA: evidence for modulation by avermectin.

Membrane currents were recorded from Xenopus laevis oocytes injected with C. elegans poly(A)+ RNA. In such oocytes glutamate activated an inward membrane current that desensitized in the continued presence of glutamate. Glutamate-receptor agonists quisqualate, kainate, and N-methyl-D-aspartate were inactive. The reversal potential of the glutamate-sensitive current was -22 mV, and exhibited a strong dependence on external chloride with a 48 mV change for a 10-fold change in chloride. The chloride channel blockers flufenamate and picrotoxin inhibited the glutamate-sensitive current. Ibotenate, a structural analog of glutamate, also activated a picrotoxin-sensitive chloride current. Ibotenate was inactive when current was partially desensitized with glutamate, and the responses to low concentrations of glutamate and ibotenate were additive. The anthelmintic/insecticide compound avermectin directly activated the glutamate-sensitive current. In addition, avermectin increased the response to submaximal concentrations of glutamate, shifted the glutamate concentration-response curve to lower concentrations, and slowed the desensitization of glutamate-sensitive current. We propose that the glutamate-sensitive chloride current and the avermectin-sensitive chloride current are mediated via the same channel.

Animals↗
PubMed1986

The activity of non-steroidal anti-inflammatory drugs in the rat mesenteric vasculature.

Rat isolated, perfused mesenteric blood vessels have been used to determine whether non-steroidal anti-inflammatory drugs (NSAIDs) display selectivity for inhibition of vasoconstrictor responses to noradrenaline compared with those to calcium. The rank order of potency of the NSAIDs for inhibition of responses to noradrenaline was: meclofenamate greater than flufenamate = diclofenac greater than indomethacin greater than fenbufen greater than phenylbutazone greater than ibuprofen greater than ketoprofen greater than naproxen greater than paracetamol (included as an inhibitor of cyclo-oxygenase). All NSAIDs show selectivity for noradrenaline (mean selectivity molar ratio = 0.19; s.e.m. 0.15-0.23) but there was a positive correlation (r = 0.98, P less than 0.001) between inhibition of responses to noradrenaline and those to calcium. The depressant effect of meclofenamate and of fenbufen, the most potent and most selective in the series, respectively, on responses to noradrenaline, was completely reversed to control values by prostaglandin E2. The results support previous findings that inhibition of cyclo-oxygenase in rat mesenteric blood vessels leads to a loss of response to noradrenaline. Comparison of the present data for inhibition of responses to noradrenaline in the mesentery with published data indicates that the mesentery bears a greater similarity to other in-vitro rather than in-vivo models for screening NSAIDs.

Animals↗
PubMed1990

The aspirin and heme-binding sites of ovine and murine prostaglandin endoperoxide synthases.

Acetylation of Ser-530 of sheep prostaglandin endoperoxide (PGG/H) synthase by aspirin causes irreversible inactivation of the cyclooxygenase activity of the enzyme. To determine the catalytic function of the hydroxyl group of Ser-530, we used site-directed mutagenesis to replace Ser-530 with an alanine. Cos-1 cells transfected with expression vectors containing the native (Ser-530) or mutant (Ala-530) cDNAs for sheep PGG/H synthase expressed comparable cyclooxygenase and hydroperoxidase activities. Km values for arachidonate (8 microM) and ID50 values for reversible inhibition by the cyclooxygenase inhibitors, flurbiprofen (5 microM), flufenamate (20 microM), and aspirin (20 mM), were also the same for both native and mutant PGG/H synthases; however, only the native enzyme was irreversibly inactivated by aspirin. Thus, the "active site" Ser-530 of PGG/H synthase is not essential for catalysis or substrate binding. Apparently, acetylation of native PGG/H synthase by aspirin introduces a bulky sidechain at position 530 which interferes with arachidonate binding. In related studies, a cDNA for mouse PGG/H synthase was cloned and sequenced. A sequence of 35 residues with Ser-530 at the midpoint was identical in the two proteins. Thus, Ser-530 does lie in a highly conserved region, probably involved in cyclooxygenase catalysis. Sequence comparisons of mouse and sheep PGG/H synthase also provided information about the heme-binding site of the enzyme. The sheep HYPR sequence (residues 274-277), which had been proposed to form a portion of the distal heme-binding site, is not conserved in the mouse PGG/H synthase, suggesting that this region is not the distal heme-binding site. One sequence, TIWLREHNRV (residues 303-312 of the sheep enzyme), is very closely related to the sequence TLW(L)LREHNRL common to thyroid peroxidase and myeloperoxidase. The histidine in this latter sequence is the putative axial heme ligand of these peroxidases. We suggest that the histidine (His-309) of sheep PGG/H synthase sequence is the axial heme ligand of this enzyme.

Amino Acid Sequence↗