Activation of Calcium- and Voltage-gated Potassium Channels of Large Conductance by Leukotriene B4

Calcium/voltage-gated, large conductance potassium (BK) channels control numerous physiological processes, including myogenic tone. BK channel regulation by direct interaction between lipid and channel protein sites has received increasing attention. Leukotrienes (LTA4, LTB4, LTC4, LTD4, and LTE4) are inflammatory lipid mediators. We performed patch clamp studies in Xenopus oocytes that co-expressed BK channel-forming (cbv1) and accessory β1 subunits cloned from rat cerebral artery myocytes. Leukotrienes were applied at 0.1 nm–10 μm to either leaflet of cell-free membranes at a wide range of [Ca²⁺]_i and voltages. Only LTB4 reversibly increased BK steady-state activity (EC₅₀ = 1 nm; E_max reached at 10 nm), with physiological [Ca²⁺]_i and voltages favoring this activation. Homomeric cbv1 or cbv1-β2 channels were LTB4-resistant. Computational modeling predicted that LTB4 docked onto the cholane steroid-sensing site in the BK β1 transmembrane domain 2 (TM2). Co-application of LTB4 and cholane steroid did not further increase LTB4-induced activation. LTB4 failed to activate β1 subunit-containing channels when β1 carried T169A, A176S, or K179I within the docking site. Co-application of LTB4 with LTA4, LTC4, LTD4, or LTE4 suppressed LTB4-induced activation. Inactive leukotrienes docked onto a portion of the site, probably preventing tight docking of LTB4. In summary, we document the ability of two endogenous lipids from different chemical families to share their site of action on a channel accessory subunit. Thus, cross-talk between leukotrienes and cholane steroids might converge on regulation of smooth muscle contractility via BK β1. Moreover, the identification of LTB4 as a highly potent ligand for BK channels is critical for the future development of β1-specific BK channel activators.     

Regulation of the Temperature-dependent Activation of Transient Receptor Potential Vanilloid 1 (TRPV1) by Phospholipids in Planar Lipid Bilayers

TRPV1 (transient receptor potential vanilloid 1) proteins are heat-activated nonselective cation channels. TRPV1 channels are polymodal in their function and exhibit multifaceted regulation with various molecular compounds. In this regard, phosphoinositides, particularly phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate, are important channel regulators. However, their effects on TRPV1 channel activity have not been conclusively determined. To characterize temperature-induced activation of TRPV1 in the presence of different phospholipids, we purified the TRPV1 protein from HEK-293 cells and incorporated it into planar lipid bilayers. In the presence of 2.5 μm phosphatidylinositol 4,5-bisphosphate, TRPV1 channels demonstrated rapid activation at 33–39 °C and achieved full channel opening at 42 °C. At this temperature range, TRPV1 heat activation exhibited steep temperature dependence (temperature coefficient (Q₁₀) of 18), and the channel openings were accompanied by large changes in entropy and enthalpy, suggesting a substantial conformation change. At a similar temperature range, another phosphoinositide, phosphatidylinositol 4-phosphate, also potentiated heat activation of TRPV1, but with much lower efficiency. Negatively charged phosphatidylglycerol could also induce heat activation of TRPV1 channels, although with a small-conductance state. Our data demonstrate that phospholipids, specifically phosphoinositides, are important regulators of TRPV1 and are required for heat-induced channel activity.     

Modulation of the Voltage-gated Potassium Channel (Kv4.3) and the Auxiliary Protein (KChIP3) Interactions by the Current Activator NS5806

KChIP3 (potassium channel interacting protein 3) is a calcium-binding protein that binds at the N terminus of the Kv4 voltage-gated potassium channel through interactions at two contact sites and has been shown to regulate potassium current gating kinetics as well as channel trafficking in cardiac and neuronal cells. Using fluorescence spectroscopy, isothermal calorimetry, and docking simulations we show that the novel potassium current activator, NS5806, binds at a hydrophobic site on the C terminus of KChIP3 in a calcium-dependent manner, with an equilibrium dissociation constant of 2–5 μm in the calcium-bound form. We further determined that the association between KChIP3 and the hydrophobic N terminus of Kv4.3 is calcium-dependent, with an equilibrium dissociation constant in the apo-state of 70 ± 3 μm and 2.7 ± 0.1 μm in the calcium-bound form. NS5806 increases the affinity between KChIP3 and the N terminus of Kv4.3 (K_d = 1.9 ± 0.1 μm) in the presence and absence of calcium. Mutation of Tyr-174 or Phe-218 on KChIP3 abolished the enhancement of Kv4.3 site 1 binding in the apo-state, highlighting the role of these residues in drug and K4.3 binding. Kinetic studies show that NS5806 decreases the rate of dissociation between KChIP3 and the N terminus of K_V4.3. Overall, these studies support the idea that NS5806 directly interacts with KChIP3 and modulates the interactions between this calcium-binding protein and the T1 domain of the Kv4.3 channels through reorientation of helix 10 on KChIP3.     

Translocation of iron citrate and phosphorus in xylem exudate of soybean

Soybean plants, Glycine max (L.) Merrill, in standard solution received 2.5 μm ferric ethylenediamine di(o-hydroxyphenylacetate (FeEDDHA) and 0 to 128 μm phosphorus. Their stem exudates contained: 32 to 52 μm Fe, 120 to 5000 μm P, and 120 to 165 μm citrate. Electrophoresis of exudates with high P caused Fe trailing that precluded identification of any major form of Fe. Exudate with low P gave an anodic band of Fe citrate as the major Fe compound. Phosphate added to exudate in vitro depressed the Fe citrate peak and cause Fe trailing. EDDHA added to exudate in vitro pulled Fe from Fe citrate; citrate then migrated as a slower form and Fe migrated as FeEDDHA. A modified preculture system, involving 2-day renewals of 0.2 μm FeEDDHA with 3.2, 9.6, or 16 μm P and low levels of other ions, controlled pH depression and produced considerable change in citrate and P levels. The exudates contained: 45 to 57 μm Fe, 200 to 925 μm P, and 340 to 1025 μm citrate. The high citrate was from plants grown with low P. The major form of Fe in the exudates was Fe citrate. This is probably the form translocated in the plants.     

Interaction Between Potassium and Calcium in Their Absorption by Intact Barley Plants. II. Effects of Calcium and Potassium Concentration on Potassium Absorption

Increasing concentrations of K (20, 200, 2000 μm) in the nutrient solution depressed Ca content and concentration in barley plants growing in nutrient solutions of low Ca concentrations (250 and 2500 μm). Increasing K from 20 to 200 μm depressed Ca absorption more than increasing K from 200 to 2000 μm K.     

Effects of Cycloheximide on Indoleacetic Acid-induced Ethylene Production in Pea Root Tips

Cycloheximide inhibited ethylene production in excised pea root tips treated with high levels of indoleacetic acid (100 μm and 10 μm). In contrast, cycloheximide did not inhibit ethylene production induced by a lower concentration (1 μm) of indoleacetic acid unless it was added 2 hours before the indoleacetic acid treatment. These observations suggest that indoleacetic acid has two effects on the enzyme system involved in ethylene synthesis. At low concentrations (1 μm) indoleacetic acid increases ethylene production without protein synthesis, whereas at the higher concentrations, the synthesis of new protein is associated with increased ethylene production.     

Sequential Induction of Phenylalanine Ammonia-lyase and a Lyase-inactivating System in Potato Tuber Disks

The light induced synthesis of phenylalanine ammonia-lyase in disks cut from potato tubers is very sensitive to cycloheximide. Synthesis is inhibited 50% in disks cultured on 5 μm cycloheximide instead of water and almost completely in disks aged in the presence of 10 μm inhibitor. Inhibition is irreversible. Fresh disks exposed only 1 hour to 10 μm cycloheximide do not synthesize enzyme during the subsequent 24 hours.     

Inhibition of chloroplast reactions with phenylmercuric acetate

Phenylmercuric acetate is a selective inhibitor of the photosynthetic activities of isolated spinach (Spinacia oleracea) chloroplasts. At 5 μm concentration of phenylmercuric acetate, photophosphorylation is inhibited. At 33 μm phenylmercuric acetate, ferredoxin is inactivated. Ferredoxin-NADP oxidoreductase is 50% inhibited at 100 μm phenylmercuric acetate. Photosystem II reactions are 50% inhibited at 150 μm phenylmercuric acetate and very much higher cooncentrations—500 μm—are needed to approach complete inhibition. Phenylmercuric acetate inhibition of photosystem II appears to be selective, blocking a site between the 3-(3,4-dichlorophenyl)-1,1-dimethyl urea sensitive site and the site inactivated by high concentrations of tris buffer.     

Photoreduction and Photophosphorylation with Tris-Washed Chloroplasts

The artificial electron donor compounds p-phenylenediamine (PD), N, N, N′, N′-tetramethyl-p-phenylenediamine (TMPD), and 2,6-dichlorophenol-indophenol (DCPIP) restored the Hill reaction and photophosphorylation in chloroplasts that had been inhibited by washing with 0.8 m tris (hydroxymethyl) aminomethane (tris) buffer, pH 8.0. The tris-wash treatment inhibited the electron transport chain between water and photosystem II and electron donation occurred between the site of inhibition and photosystem II. Photoreduction of nicotinamide adenine dinucleotide phosphate (NADP) supported by 33 μm PD plus 330 μm ascorbate was largely inhibited by 1 μm 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) while that supported by 33 μm TMPD or DCPIP plus ascorbate was relatively insensitive to DCMU. Experiments with the tris-washed chloroplasts indicated that electron donors preferentially donate electrons to photosystem II but in the presence of DCMU the donors (with the exception of PD at low concentrations) could also supply electrons after the DCMU block. The PD-supported photoreduction of NADP showed the relative inefficiency in far-red light characteristic of chloroplast reactions requiring photosystem II. With phosphorylating systems involving electron donors at low concentrations (33 μm donor plus 330 μm ascorbate) photophosphorylation, which occurred with P/e₂ ratios approaching unity, was completely inhibited by DCMU but with higher concentrations of the donor systems, photophosphorylation was only partially inhibited.     

Activation of pyruvate dehydrogenase in perfused rat heart by dichloroacetate (Short Communication)

The activity of pyruvate dehydrogenase was assayed in extracts of rat hearts perfused in vitro with media containing glucose and insulin±acetate±dichloroacetate. Dichloroacetate (100μm, 1mm or 10mm) increased the activity of pyruvate dehydrogenase in perfusions with glucose or glucose+acetate. Evidence is given that dichloroacetate may facilitate the conversion of pyruvate dehydrogenase from an inactive (phosphorylated) form into an active (dephosphorylated) form.     

Serine Transhydroxymethylase of Cauliflower (Brassica oleracea var. botrytis L.): Partial Purification and Properties

Serine transhydroxymethylase (EC 2.1.2.1) has been purified 46-fold from cauliflower (Brassica oleracea var. botrytis L.). The enzyme was completely dependent on the presence of tetrahydrofolic acid for the conversion of serine to glycine. The addition of pyridoxal phosphate gave a large increase in the reaction rate. A double pH optimum was observed with maxima at 7.5 and 9.5. The enzyme is specific for l-serine. The d-isomer is neither a substrate nor an inhibitor. The Michaelis constants for l-serine, tetrahydrofolic acid, and pyridoxal phosphate were 300 μm, 760 μm, and 24 μm, respectively. The addition of K⁺ also stimulated the reaction rate considerably. The effect was quite specific since all other metal ions tested either had very little: influence or were extremely inhibitory.     

Stable changes to calcium fluxes in mitochondria isolated from rat livers perfused with α-adrenergic agonists and with glucagon

1. Human uterine cervical stroma was found to contain a Ca²⁺-independent neutral proteinase against casein and N-benzoyl-dl-arginine p-nitroanilide (Bz-dl-Arg-Nan). This enzyme was tightly bound to an insoluble material (20000g pellet) and was solubilized by high concentrations of NaCl or KCl. High concentrations of them in the reaction system, however, inhibited reversibly the activity of this enzyme. 2. The neutral proteinase was partially purified by extraction with NaCl, gel filtration on Sephadex G-200 and affinity chromatography on casein–Sepharose. 3. The optimal pH of this partially purified enzyme was 7.4–8.0 against casein and Bz-dl-Arg-Nan. The molecular weight of the enzyme was found to be about 1.4×10⁵ by gel filtration on Sephadex G-200. 4. The enzyme was significantly inhibited by di-isopropyl phosphorofluoridate (0.1mm). High concentration of phenylmethanesulphonyl fluoride (5mm), 7-amino-1-chloro-3-l-tosylamidoheptan-2-one (0.5mm), antipain (10μm) or leupeptin (10μm) was also found to be inhibitory, but chymostatin (40μg/ml), soya-bean trypsin inhibitor (2.5mg/ml), human plasma (10%, v/v), p-chloromercuribenzoate (1mm), EDTA (10mm) and 1-chloro-4-phenyl-3-l-tosylamidobutan-2-one (1mm) had no effect on the enzyme. 5. The neutral proteinase hydrolysed casein, Bz-dl-Arg-Nan and heat-denatured collagen, but was inactive towards native collagen and several synthetic substrates, such as 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg, 3-carboxypropionyl-Ala-Ala-Ala p-nitroanilide and 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, and also proteoglycan. The enzyme did not act as a plasminogen activator. 6. These properties suggested that a neutral proteinase in the human uterine cervix was different from enzymes previously reported.     

Control of logarithmic growth rates of tobacco callus tissue by cytokinins

The growth rates of tobacco callus tissues on media containing 10⁻⁶ to 10 μm 6-(γ,γ-dimethylallylamino)purine (2iP) were measured. At concentrations of 10⁻⁴ μm and above growth rates were exponential and dependent on cytokinin concentration. At 2iP concentrations of 10⁻⁴ to 0.33 μm, the exponential rate was maintained for 4 to 5 doublings of fresh and dry weight. After this period a linear phase, resulting in approximately 1 doubling of weight, occurred. The growth of tissues on media containing higher than 0.33 μm 2iP was exponential for only about 15 days. At the end of this time, and well before they achieved half their final weight, they exhibited growth which was less rapid than logarithmic but more rapid than linear. Comparisons with zeatin, 6-benzylaminopurine and kinetin indicated that, although the maximum growth rates obtained with relatively high concentrations (0.1-1 μm) were similar, the naturally occurring cytokinins, 2iP and zeatin, promoted faster rates at lower concentrations (10⁻³-10⁻² μm) than did 6-benzylaminopurine and kinetin.     

Effects of dopamine on prolactin secretion and cyclic AMP accumulation in the rat anterior pituitary gland

The effects of dopamine on pituitary prolactin secretion and pituitary cyclic AMP accumulation were studied by using anterior pituitary glands from adult female rats, incubated in vitro. During 2h incubations, significant inhibition of prolactin secretion was achieved at concentrations between 1 and 10nm-dopamine. However, 0.1–1μm-dopamine was required before a significant decrease in pituitary cyclic AMP content was observed. In the presence of 1μm-dopamine, pituitary cyclic AMP content decreased rapidly to reach about 75% of the control value within 20min and there was no further decrease for at least 2h. Incubation with the phosphodiesterase inhibitors theophylline (8mm) or isobutylmethylxanthine (2mm) increased pituitary cyclic AMP concentrations 3- and 6-fold respectively. Dopamine (1μm) had no effect on the cyclic AMP accumulation measured in the presence of theophylline, but inhibited the isobutylmethylxanthine-induced increase by 50%. The dopamine inhibition of prolactin secretion was not affected by either inhibitor. Two derivatives of cyclic AMP (dibutyryl cyclic AMP and 8-bromo cyclic AMP) were unable to block the dopamine (1μm) inhibition of prolactin secretion, although 8-bromo cyclic AMP (2mm) significantly stimulated prolactin secretion and both compounds increased somatotropin (growth hormone) release. Cholera toxin (3μg/ml for 4h) increased pituitary cyclic AMP concentrations 4–5-fold, but had no effect on prolactin secretion. The inhibition of prolactin secretion by dopamine was unaffected by cholera toxin, despite the fact that dopamine had no effect on the raised pituitary cyclic AMP concentration caused by this factor. Dopamine had no significant effect on either basal or stimulated somatotropin secretion under any of the conditions tested. We conclude that the inhibitory effects of dopamine on prolactin secretion are probably not mediated by lowering of cyclic AMP concentration, although modulation of the concentration of this nucleotide in some other circumstances may alter the secretion of the hormone.     

Amino Acid Derivatives as Bitter Taste Receptor (T2R) Blockers

In humans, the 25 bitter taste receptors (T2Rs) are activated by hundreds of structurally diverse bitter compounds. However, only five antagonists or bitter blockers are known. In this study, using molecular modeling guided site-directed mutagenesis, we elucidated the ligand-binding pocket of T2R4. We found seven amino acids located in the extracellular side of transmembrane 3 (TM3), TM4, extracellular loop 2 (ECL2), and ECL3 to be involved in T2R4 binding to its agonist quinine. ECL2 residues Asn-173 and Thr-174 are essential for quinine binding. Guided by a molecular model of T2R4, a number of amino acid derivatives were screened for their ability to bind to T2R4. These predictions were tested by calcium imaging assays that led to identification of γ-aminobutryic acid (GABA) and Nα,Nα-bis(carboxymethyl)-l-lysine (BCML) as competitive inhibitors of quinine-activated T2R4 with an IC₅₀ of 3.2 ± 0.3 μm and 59 ± 18 nm, respectively. Interestingly, pharmacological characterization using a constitutively active mutant of T2R4 reveals that GABA acts as an antagonist, whereas BCML acts as an inverse agonist on T2R4. Site-directed mutagenesis confirms that the two novel bitter blockers share the same orthosteric site as the agonist quinine. The signature residues Ala-90 and Lys-270 play important roles in interacting with BCML and GABA, respectively. This is the first report to characterize a T2R endogenous antagonist and an inverse agonist. The novel bitter blockers will facilitate physiological studies focused on understanding the roles of T2Rs in extraoral tissues.     

Malate dehydrogenase isoenzymes in division synchronized cultures of euglena

Sucrose density gradient centrifugation of broken cell suspensions of autotrophically grown Euglena gracilis Klebs. has allowed the separation of chloroplasts, mitochondria, and peroxisomes. Chlorophyll was taken as a marker for chloroplasts, fumarase and succinate dehydrogenase for mitochondria, and glycolate oxidoreductase for peroxisomes. Peaks of malate dehydrogenase (l-malate-NAD oxidoreductase, EC 1.1.1.37) activity were found in the mitochondrial and peroxisomal fractions. Acrylamide gel electrophoresis showed specific isoenzymes in the mitochondrial and peroxisomal fractions and a third isoenzyme in the supernatant. The mitochondrial isoenzyme which had a Km (oxaloacetate) of 30μm was inhibited by oxaloacetate concentrations above 0.17 mm, an inhibition of 50% being given by 0.9 mm oxaloacetate. The peroxisomal isoenzyme had a Km (oxaloacetate) of 24 μm, was inhibited by oxaloacetate concentrations above 0.13 mm, 50% inhibition being given by 0.25 mm oxaloacetate. Malate dehydrogenase activity in the supernatant did not show inhibition by increasing oxaloacetate concentration, the Km (oxaloacetate) being 91 μm.     

Concentration of adenosine 3':5'-cyclic monophosphate in mouse pancreatic islets measured by a protein-binding radioassay

A protein-binding radioassay for cyclic AMP was modified to detect less than 0.025pmol of the nucleotide. The method was applied to the measurement of cyclic AMP in small numbers of mouse pancreatic islets (as little as 25μg of tissue) by use of barium acetate–H₂SO₄ for deproteinization. The concentration of cyclic AMP in mouse islets incubated in media containing 3.3 or 20mm-glucose was 0.016pmol/10 islets (approx. 1μm in intracellular water). Glucose concentration (3.3 or 20mm) had no detectable effect on islet concentrations of cyclic AMP with periods of incubation or perifusion ranging from 0.5 to 60min, although insulin release rate was rapidly increased by 20mm-glucose. Caffeine (5mm) or 3-isobutyl-1-methylxanthine (1mm), which are known inhibitors of islet cyclic AMP phosphodiesterase, produced marked and rapid increases in islet cyclic AMP concentration at 3.3 or 20mm-glucose, but only enhanced the insulin release rate at the higher glucose concentration. The role of cyclic AMP in insulin release induced by glucose is discussed.     

T-2 toxin decreases logarithmic growth rates of tobacco callus tissues

T-2 toxin, a mycotoxin produced by Fusarium tricinctum, decreases logarithmic growth rates of tobacco (Nicotiana tabacum L.) pith callus tissues. Toxin concentrations as low as 0.003 μm will decrease growth rates; a concentration of 0.081 μm will halt growth completely. Additional exogenous cytokinin will reduce the inhibition by toxin only when the initial cytokinin and toxin concentrations are quite low (about 0.01 μm). When inhibited tissues are transferred to media lacking toxin, they assume the faster, control rates almost immediately. Maximal yields of tissue (yields at the point at which no sugar was detected in the medium) are not affected by toxin concentrations of 0.01 to 0.036 μm.     

Terminal Oxidases of Chlorella pyrenoidosa

In studies of the kinetics of oxygen uptake by glucose-stimulated Chlorella pyrenoidosa, two terminal oxidases could be distinguished. The cytochrome oxidase of Chlorella has a Km (O₂) of 2.1 ± 0.3 μm, while the second oxidase has a Km (O₂) of 6.7 ± 0.5 μm, and a maximum capacity about one-quarter of that of the cytochrome system. The identity of the second oxidase is unknown, but it is not inhibited by carbon monoxide, 1 mm cyanide, 0.1 mm thiocyanate, or 1 mm 8-hydroxyquinoline. In fresh cultures, the second oxidase accounts for at most 35% of the total oxygen uptake.