Identification of High-Affinity Slow Anion Channel Blockers and Evidence for Stomatal Regulation by Slow Anion Channels in Guard Cells

Slow anion channels in the plasma membrane of guard cells have been suggested to constitute an important control mechanism for long-term ion efflux, which produces stomatal closing. Identification of pharmacological blockers of these slow anion channels is instrumental for understanding plant anion channel function and structure. Patch clamp studies were performed on guard cell protoplasts to identify specific extracellular inhibitors of slow anion channels. Extracellular application of the anion channel blockers NPPB and IAA-94 produced a strong inhibition of slow anion channels in the physiological voltage range with half inhibition constants (K1/2) of 7 and 10 [mu]M, respectively. Single slow anion channels that had a high open probability at depolarized potentials were identified. Anion channels had a main conductance state of 33 [plus or minus] 8 pS and were inhibited by IAA-94. DIDS, which has been shown to be a potent blocker of rapid anion channels in guard cells (K1/2 = 0.2 [mu]M), blocked less than 20% of peak slow anion currents at extracellular or cytosolic concentrations of 100 [mu]M. The pharmacological properties of slow anion channels described here differ from those recently described for rapid anion channels in guard cells, fortifying the finding that two highly distinct types or modes of voltage- and second messenger-dependent anion channel currents coexist in the guard cell plasma membrane. Bioassays using anion channel blockers provide evidence that slow anion channel currents play a substantial role in the regulation of stomatal closing. Interestingly, slow anion channels may also function as a negative regulator during stomatal opening under the experimental conditions applied here. The identification of specific blockers of slow anion channels reported here permits detailed studies of cell biological functions, modulation, and structural components of slow anion channels in guard cells and other higher plant cells.     

Increased Salt and Drought Tolerance by D-Ononitol Production in Transgenic Nicotiana tabacum L

A cDNA encoding myo-inositol O-methyltransferase (IMT1) has been transferred into Nicotiana tabacum cultivar SR1. During drought and salt stress, transformants (I5A) accumulated the methylated inositol D-ononitol in amounts exceeding 35 [mu]mol g-1 fresh weight In I5A, photosynthetic CO2 fixation was inhibited less during salt stress and drought, and the plants recovered faster than wild type. One day after rewatering drought-stressed plants, I5A photosynthesis had recovered 75% versus 57% recovery with cultivar SR1 plants. After 2.5 weeks of 250 mM NaCl in hydroponic solution, I5A fixed 4.9 [plus or minus] 1.4 [mu]mol CO2 m-2 s-1, whereas SR1 fixed 2.5 [plus or minus] 0.6 [mu]mol CO2 m-2 s-1. myo-Inositol, the substrate for IMT1, increases in tobacco under stress. Preconditioning of I5A plants in 50 mM NaCl increased D-ononitol amounts and resulted in increased protection when the plants were stressed subsequently with 150 mM NaCl. Pro, Suc, Fru, and Glc showed substantial diurnal fluctuations in amounts, but D-ononitol did not. Plant transformation resulting in stress-inducible, stable solute accumulation appears to provide better protection under drought and salt-stress conditions than strategies using osmotic adjustment by metabolites that are constitutively present.     

Acetylene Reduction by Symbiosomes and Free Bacteroids from Broad Bean (Vicia faba L.) Nodules (Role of Oxalate)

We report the presence of oxalate in the organic acid fraction of broad bean (Vicia faba L.) nodule cytosol. Using both high-performance liquid chromatography and enzymic assays, high levels of oxalate were detected (70.4 [plus or minus] 2.4 mM). To study the potential role of oxalate as an energy-yielding substrate for nitrogenase activity, free bacteroids were isolated from nodules and found to oxidize oxalate in support of C2H2 reduction under O2 tensions that were lower than those required to oxidize succinate, another dicarboxylate commonly detected in legume nodules. Symbiosomes of broad bean, isolated for the first time from amide-producing nodules, were provided with [14C]oxalate and found to have uptake kinetics with a lower affinity [Km(oxalate) = 330 [mu]M] than that for free bacteroids [Km(oxalate) = 130 [mu]M]. In anaerobic preparations of symbiosomes supplied with purified oxyleghemoglobin, O2 consumption was stimulated by oxalate from 20.2 [plus or minus] 0.8 nmol O2 min-1mg-1 protein to 24.5 [plus or minus] 1.1 nmol O2 min-1 mg-1 protein but always remained lower than the rate of O2 consumption in free bacteroids (32.2 [plus or minus] 1.4 nmol O2 min-1 mg-1 protein). Under these conditions, C2H2 reduction activity was 9.7 [plus or minus] 0.8 and 15.1 [plus or minus] 0.9 nmol C2H4 min-1 mg-1 protein for symbiosomes and bacteroids, respectively. These data support the suggestion that oxalate may play a role as a carbon substrate in support of N2 fixation in broad bean nodules.     

Diffusional water permeability of human erythrocytes and their ghosts

The diffusional water permeability of human red cells and ghosts was determined by measuring the rate of tracer efflux by means of an improved version of the continuous flow tube method, having a time resolution of 2-3 ms. At 25 degrees C, the permeability was 2.4 x 10(3) and 2.9 x 10(3) cm s-1 for red cells and ghosts, respectively. Permeability was affected by neither a change in pH from 5.5 to 9.5, nor by osmolality up to 3.3 osmol. Manganous ions at an extracellular concentration of 19 mM did not change diffusional water permeability, as recently suggested by NMR measurements. A "ground" permeability of 1 x 10(3) cm s-1 was obtained by inhibition with 1 mM of either p- chloromercuribenzoate (PCMB) or p-chloromercuribenzene sulfonate (PCMBS). Inhibition increased temperature dependence of water permeability for red cells and ghosts from 21 to 30 kJ mol-1 to 60 kJ mol-1. Although diffusional water permeability is about one order of magnitude lower than osmotic permeability, inhibition with PCMB and PCMBS, temperature dependence both before and after inhibition, and independence of osmolality showed that diffusional water permeability has qualitative features similar to those reported for osmotic permeability, which indicates that the same properties of the membrane determine both types of transport. It is suggested that the PCMB(S)- sensitive permeability above the ground permeability takes place through the intermediate phase between integral membrane proteins and their surrounding lipids.     

Intermolecular charge transfer involving tryptophan, tyrosine and three electron-bonded intermediates derived from methionine

Oxidation processes of radiation-generated three-electron-bonded intermediates derived from methionine Met2[S+...S] and Met[S...X] (X=Cl,Br) were investigated through reaction with tryptophan and tyrosine, using the optical pulse radiolysis method. Bimolecular rate constants have been measured for the reactions Met2[S+...S] with TrpH(k=3.8 x 10(8) dm3 mol-1 s-1 and k = 4.9 X 10(8) dm3 mol-1 s-1 at at ph 1 and 4.3, respectively) and Met2[S+...S] with tyrosine, k=3.8 x 10(7) dm3 mol-1 s-1. Rate constants for intermolecular transformation of Met[S...Br] and Met[S...Cl] into TrpH+. or Trp. were also estimated. They varied from 4.7 X 10(8) dm3 mol-1 s-1 (bromide species) to 1.0 X 10(9)dm3 mol-1 s-1 (chloride species). It has also been established azide radicals N-6, N.3 in contrast to dihalide radicals (X-2) do not form transients of Met[S...X] (X = N3)-type. However, oxidation of N-3 by Met2[S+...S] occurs with a bimolecular rate constant of 2.8 X 10(8) dm3 mol-1 s-1. These results are discussed in the light of some equilibria which have been proposed earlier for methionine-halide systems.     

Urea permeability of human red cells

The rate of unidirectional [14C]urea efflux from human red cells was determined in the self-exchange and net efflux modes with the continuous flow tube method. Self-exchange flux was saturable and followed simple Michaelis-Menten kinetics. At 38 degrees C the maximal self-exchange flux was 1.3 X 10(-7) mol cm-2 s-1, and the urea concentration for half-maximal flux, K1/2, was 396 mM. At 25 degrees C the maximal self-exchange flux decreased to 8.2 X 10(-8) mol cm-2 s-1, and K1/2 to 334 mM. The concentration-dependent urea permeability coefficient was 3 X 10(-4) cm s-1 at 1 mM and 8 X 10(-5) cm s-1 at 800 mM (25 degrees C). The latter value is consonant with previous volumetric determinations of urea permeability. Urea transport was inhibited competitively by thiourea; the half-inhibition constant, Ki, was 17 mM at 38 degrees C and 13 mM at 25 degrees C. Treatment with 1 mM p-chloromercuribenzosulfonate inhibited urea permeability by 92%. Phloretin reduced urea permeability further (greater than 97%) to a "ground" permeability of approximately 10(-6) cm s-1 (25 degrees C). This residual permeability is probably due to urea permeating the hydrophobic core of the membrane by simple diffusion. The apparent activation energy, EA, of urea transport after maximal inhibition was 59 kJ mol-1, whereas in control cells EA was 34 kJ mol-1 at 1 M and 12 kJ mol-1 at 1 mM urea. In net efflux experiments with no extracellular urea, the permeability coefficient remained constantly high, independent of a variation of intracellular urea between 1 and 500 mM, which indicates that the urea transport system is asymmetric. It is concluded that urea permeability above the ground permeability is due to facilitate diffusion and not to diffusion through nonspecific leak pathways as suggested previously.     

The Mechanism of Amino Acid Efflux from Seed Coats of Developing Pea Seeds as Revealed by Uptake Experiments

The uptake of amino acids by excised seed coat halves of developing seeds of pea (Pisum sativum L.) was characterized. The influx of L-valine and L-glutamic acid was proportional to their external concentration, with coefficients of proportionality (k) of 11.0 and 7.1 [mu]mol g-1 fresh weight min-1 M-1, respectively. The influx of L-lysine could be analyzed into a component with linear kinetics (k = 8.1 [mu]mol g-1 fresh weight min-1 M-1) and one with saturation kinetics (Michaelis constant = 6.5 mM), but the latter may have resulted from the mutual interaction between the influx of the cationic lysine and the membrane potential. The influx of the amino acids was not affected by 10 [mu]M carbonylcyanide m-chlorophenylhydrazone, but was inhibited by about 50% in the presence of 2.5 mM p-chloromercuribenzene sulfonic acid. Conservative estimates of the permeability coefficients of the plasma membrane of seed coat parenchyma cells for lysine, glutamic acid, and several neutral amino acids were all in the range of 4 x 10-7 cm s-1 to 9 x 10-7 cm s-1, which is 4 to 5 orders of magnitude greater than those reported for artificial lipid bilayers. It is concluded that nonselective pores constitute a pathway in the plasma membrane for passive transport of amino acids. It is argued that this pathway is also used for the efflux of endogenous amino acids, the process by which nitrogen becomes available for the embryo.     

Rapid [gamma]-Aminobutyric Acid Synthesis and the Inhibition of the Growth and Development of Oblique-Banded Leaf-Roller Larvae

The hypothesis that rapid [gamma]-aminobutyrate (GABA) accumulation is a plant defense against phytophagous insects was investigated. Increasing GABA levels in a synthetic diet from 1.6 to 2.6 [mu]mol g-1 fresh weight reduced the growth rates, developmental rates, and survival rates of cultured Choristoneura rosaceana cv Harris larvae. Simulation of the mechanical damage resulting from phytophagous activity increased soybean (Glycine max L.) leaf GABA 10- to 25-fold within 1 to 4 min. Pulverizing leaf tissue resulted in a value of 2.15 ([plus or minus]0.11 SE) [mu]mol GABA g-1 fresh weight.     

Osmoregulation by Oat Coleoptile Protoplasts (Effect of Auxin)

The effect of auxin on the physiology of protoplasts from growing oat (Avena sativa L.) coleoptiles was investigated. Protoplasts, isolated iso-osmotically from peeled oat coleoptile segments, were found to swell steadily over many hours. Incubated in 1 mM CaCl2, 10 mM KCl, 10 mM 2-(morpholino)ethanesulfonic acid/1,3-bis-[tris(hydroxymethyl)methylamino]propane, pH 6.5, and mannitol to 300 milliosmolal, protoplasts swelled 28.9% [plus or minus] 2.0 (standard error) after 6 h. Addition of 10 [mu]M indoleacetic acid (IAA) increased swelling to 41.1% [plus or minus] 2.1 (standard error) after 6 h. Swelling (in the absence of IAA) was partially dependent on K+ in the bath medium, whereas auxin-induced swelling was entirely dependent on K+. Replacement of mannitol in the bath by Glc increased swelling (in the absence of IAA) and eliminated auxin-induced swelling. Swelling with or without IAA was inhibited by osmotic shock and was completely reversed by 0.1 mM NaN3. Sodium orthovanadate, applied at 0.5 mM, only gradually inhibited swelling under various conditions but was most effective with protoplasts prepared from tissue preincubated in vanadate. Our data are interpreted to suggest that IAA increases the conductance of the plasma membrane to K+.     

Water permeability and revolving movement in Phaseolus vulgaris L. twining shoots

Osmotic water permeability (Pos) was measured in protoplasts isolated from different tissues of Phaseolus vulgaris twining shoot. Parenchyma protoplasts exhibited more frequently high Pos values than epidermis protoplasts did. Water channels could facilitate water movement between parenchyma cells whereas cell-to-cell water transport mostly occurs through plasmodesmata in epidermis.     

Correction

Phosphoribosylpyrophosphate synthetase (PRS; EC 2.7.6.1) from Hevea brasiliensis Mull. Arg. latex was located in the cytosol. After purification, its apparent molecular weight under nondenaturing conditions was estimated at 200,000 [plus or minus] 10,000; a single band at 57,000 [plus or minus] 3,000 was detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme seemed to be a homotetramer. Its affinity constants were estimated at 200 [plus or minus] 30 [mu]M for adenosine triphosphate and 40 [plus or minus] 2 [mu]M for ribose-5-phosphate. The purified enzyme proved to be functional in a paraphysiological medium (cytosol deproteinized by ultrafiltration). Optimum pH was 7.5 in buffer and 6.5 in a paraphysiological medium. No PRS activity was detected in the absence of the Mg2+ ion. Of the numerous compounds tested, only Mn2+, phosphoribosylpyrophosphate and inorganic phosphate affected the enzymatic reaction. Mn2+ (inhibitor constant = 20 [mu]M) and phosphoribosylpyrophosphate (inhibitor constant = 30 [mu]M) were inhibitors. PRS responded allosterically (Hill's coefficient = 2.3) to ribose-5-phosphate in the presence of a physiological concentration of inorganic phosphate (10 mM). These results are set in the physiological context of laticifers.     

Purification and Characterization of Phosphoribosylpyrophosphate Synthetase from Rubber Tree Latex

Phosphoribosylpyrophosphate synthetase (PRS; EC 2.7.6.1) from Hevea brasiliensis Mull. Arg. latex was located in the cytosol. After purification, its apparent molecular weight under nondenaturing conditions was estimated at 200,000 [plus or minus] 10,000; a single band at 57,000 [plus or minus] 3,000 was detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme seemed to be a homotetramer. Its affinity constants were estimated at 200 [plus or minus] 30 [mu]M for adenosine triphosphate and 40 [plus or minus] 2 [mu]M for ribose-5-phosphate. The purified enzyme proved to be functional in a paraphysiological medium (cytosol deproteinized by ultrafiltration). Optimum pH was 7.5 in buffer and 6.5 in a paraphysiological medium. No PRS activity was detected in the absence of the Mg2+ ion. Of the numerous compounds tested, only Mn2+, phosphoribosylpyrophosphate, and inorganic phosphate affected the enzymatic reaction. Mn2+ (inhibitor constant = 20 [mu]M) and phosphoribosylpyrophosphate (inhibitor constant = 30 [mu]M) were inhibitors. PRS responded allosterically (Hill's coefficient = 2.3) to ribulose-5-phosphate in the presence of a physiological concentration of inorganic phosphate (10 mM). These results are set in the physiological context of laticifers.     

Composition and Distribution of Adenylates in Soybean (Glycine max L.) Nodule Tissue

Adenylates (ATP, ADP, and AMP) may play a central role in the regulation of the O2-limited C and N metabolism of soybean nodules. To be able to interpret measurements of adenylate levels in whole nodules and to appreciate the significance of observed changes in adenylates associated with changes in O2-limited metabolism, methods were developed for measuring in vivo levels of adenylate pools in the cortex, plant central zone, and bacteroid fractions of soybean (Glycine max L. Merr cv Maple Arrow x Bradyrhizobium japonicum strain USDA 16) nodules. Intact nodulated roots were either frozen in situ by flushing with prechilled Freon-113(-156[deg]C) or by rapidly (<1 s) uprooting plants and plunging them into liquid N2. The adenylate energy charge (AEC = [ATP + 0.5 x ADP]/[ATP + ADP + AMP]) of whole-nodule tissue (0.65 [plus or minus] 0.01, n = 4) was low compared to that of subtending roots (0.80 [plus or minus] 0.03, n = 4), a finding indicative of hypoxic metabolism in nodules. The cortex and central zone tissues were dissected apart in lyophilized nodules, and AEC values were 0.84 [plus or minus] 0.04 and 0.61 [plus or minus] 0.03, respectively. Although the total adenylate pool in the lyophilized nodules was only 41% of that measured in hydrated tissues, the AEC values were similar, and the lyophilized nodules were assumed to provide useful material for assessing adenylate distribution. The nodule cortex contained 4.4% of whole-nodule adenylates, with 95.6% being located in the central zone. Aqueous fractionation of bacteroids from the plant fraction of whole nodules and the use of marker enzymes or compounds to correct for recovery of bacteroids and cross-contamination of the bacteroid and plant fractions resulted in estimates that 36.2% of the total adenylate pool was in bacteroids, and 59.4% was in the plant fraction of the central zone. These are the first quantitative assessments of adenylate distribution in the plant and bacteroid fractions of legume nodules. These estimates were combined with theoretical calculations of rates of ATP consumption in the cortex (9.5 nmol g-1 fresh weight of nodule s-1), plant central zone (38 nmol g-1 fresh weight of nodule s-1), and bacteroids (62 nmol g-1 fresh weight of nodule s-1) of soybean nodules to estimate the time constants for turnover of the total adenylate pool and the ATP pool within each nodule fraction. The low values for time constant (1.6-5.8 s for total adenylate, 0.9-2.5 s for ATP only) in each fraction reflect the high metabolic activity of soybean nodules and provide a background for further studies of the role of adenylates in O2-limited nodule metabolism.     

Functional colocalization of water channels and proton pumps in endosomes from kidney proximal tubule

The apical membrane of mammalian proximal tubule undergoes rapid membrane cycling by exocytosis and endocytosis. Osmotic water and ATP- driven proton transport were measured in endocytic vesicles from rabbit and rat proximal tubule apical membrane labeled in vivo with the fluid phase marker fluorescein-dextran. Osmotic water permeability (Pf) was determined from the time course of fluorescein-dextran fluorescence after exposure of endosomes to an inward osmotic gradient in a stopped- flow apparatus. Pf was 0.009 (rabbit) and 0.029 cm/s (rat) (23 degrees C) and independent of osmotic gradient size. Pf in rabbit endosomes was inhibited reversibly by HgCl2 (KI = 0.2 mM) and had an activation energy of 6.4 +/- 0.5 kcal/mol (15-35 degrees C). Endosomal proton ATPase activity was measured from the time course of internal pH, measured by fluorescein-dextran fluorescence, after the addition of external ATP. Endosomes contained an ATP-driven proton pump that was sensitive to N-ethylmaleimide and insensitive to vanadate and oligomycin. In response to saturating [ATP] the pump acidified the endosomal compartment at a rate of 0.17 (rat) and 0.029 pH unit/s (rabbit); at an external pH of 7.4, the steady-state pH was 6.4 (rat) and 6.5 (rabbit). To examine whether water channels and the proton ATPase were present in the same endosome, the time course of fluorescein-dextran fluorescence was measured in response to an osmotic gradient in the presence and absence of ATP. ATP did not alter endosome Pf, but decreased the amplitude of the fluorescence signal by 43 +/- 3% (rabbit) and 47 +/- 4% (rat).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative permeability of canine visceral and parietal pleura

To determine the permeability of canine pleural mesothelium, visceral and intercostal parietal pleura from mongrel dogs was carefully stripped from the underlying tissue and mounted as a planar sheet in a Ussing-type chamber. The hydraulic conductivity (Lp) was determined from the rate of volume flux in response to hydrostatic pressure gradients applied to either the mucosal or serosal surface of the pleural membrane. The diffusional permeability (Pd) of radiolabeled water, sucrose, inulin, and albumin was determined under equilibrium conditions from the unidirectional tracer flux. The Lp of the visceral pleura was 0.39 +/- 0.032 (SE) X 10(-4) ml.s-1.cmH2O-1.cm-2 and that Lp of parietal pleura was 1.93 +/- 0.93 X 10(-4) ml.s-1.cmH2O-1.cm-2 (P less than 0.001). The Pd of the visceral pleura ranged from 12.21 +/- 0.45 X 10(-4) cm/s for 3H2O to 0.34 +/- 0.03 X 10(-4) cm/s for [3H]albumin. The Pd of the parietal pleura for water and sucrose was similar to that of the visceral membrane, whereas its Pd for the larger inulin and albumin molecules was greater than that of visceral pleura (P less than 0.01). A spontaneous potential difference could not be detected across either membrane. The relatively higher parietal pleural Lp and Pd for larger solutes is probably due to the presence of stomata in this membrane. These results indicate that both the parietal and the visceral pleura are extremely permeable tissues which offer little resistance to water and solute flux.     

Transient transcapillary exchange of water driven by osmotic forces in the heart

Osmotic transient responses in organ weight after changes in perfusate osmolarity have implied steric hindrance to small-molecule transcapillary exchange, but tracer methods do not. We obtained osmotic weight transient data in isolated, Ringer-perfused rabbit hearts with NaCl, urea, glucose, sucrose, raffinose, inulin, and albumin and analyzed the data with a new anatomically and physicochemically based model accounting for 1) transendothelial water flux, 2) two sizes of porous passages across the capillary wall, 3) axial intracapillary concentration gradients, and 4) water fluxes between myocytes and interstitium. During steady-state conditions approximately 28% of the transcapillary water flux going to form lymph was through the endothelial cell membranes [capillary hydraulic conductivity (Lp) = 1.8 +/- 0.6 x 10-8 cm. s-1. mmHg-1], presumably mainly through aquaporin channels. The interendothelial clefts (with Lp = 4.4 +/- 1.3 x 10-8 cm. s-1. mmHg-1) account for 67% of the water flux; clefts are so wide (equivalent pore radius was 7 +/- 0.2 nm, covering approximately 0.02% of the capillary surface area) that there is no apparent hindrance for molecules as large as raffinose. Infrequent large pores account for the remaining 5% of the flux. During osmotic transients due to 30 mM increases in concentrations of small solutes, the transendothelial water flux was in the opposite direction and almost 800 times as large and was entirely transendothelial because no solute gradient forms across the pores. During albumin transients, gradients persisted for long times because albumin does not permeate small pores; the water fluxes per milliosmolar osmolarity change were 200 times larger than steady-state water flux. The analysis completely reconciles data from osmotic transient, tracer dilution, and lymph sampling techniques.     

Role of the Plasma Membrane H+-ATPase in K+ Transport

The role of the plant plasma membrane H+-ATPase in K+ uptake was examined using red beet (Beta vulgaris L.) plasma membrane vesicles and a partially purified preparation of the red beet plasma membrane H+-ATPase reconstituted in proteoliposomes and planar bilayers. For plasma membrane vesicles, ATP-dependent K+ efflux was only partially inhibited by 100 [mu]M vanadate or 10 [mu]M carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. However, full inhibition of ATP-dependent K+ efflux by these reagents occurred when the red beet plasma membrane H+-ATPase was partially purified and reconstituted in proteoliposomes. When reconstituted in a planar bilayer membrane, the current/voltage relationship for the plasma membrane H+-ATPase showed little effect of K+ gradients imposed across the bilayer membrane. When taken together, the results of this study demonstrate that the plant plasma membrane H+-ATPase does not mediate direct K+ transport chemically linked to ATP hydrolysis. Rather, this enzyme provides a driving force for cellular K+ uptake by secondary mechanisms, such as K+ channels or H+/K+ symporters. Although the presence of a small, protonophore-insensitive component of ATP-dependent K+ transport in a plasma membrane fraction might be mediated by an ATP-activated K+ channel, the possibility of direct K+ transport by other ATPases (i.e. K+-ATPases) associated with either the plasma membrane or other cellular membranes cannot be ruled out.     

Identification of the factors affecting the rate of deactivation of hypochlorous acid by melatonin

It has been found that melatonin reacts rapidly with hypochlorous acid in phosphate-buffered, ethanol-water solutions to produce 2-hydroxymelatonin. The rate law, d[2 - HOMel]/dt - kHOCl[Mel][HOCl] - kOCl-[Mel][OCl-], was obtained. At 37 degrees C and at a water concentration of 23.5 M, kOCl- = 6.0 x 10(2) L. mol-1. s-1, and kHOCl was found to be a function of the water concentration, kHOCl = 11 +/- 3 L3. mol-3. s-1. [H2O]2, indicating that the availability of water at the site of the reaction plays a significant role. The part that the structural components of melatonin play in determining the reaction pathway was examined by comparing the rate of deactivation of HOCl by melatonin to that of the model compounds indole, 5-methoxyindole, and 3-methylindole. The relative reactivity is explained in terms of steric and electronic effects, and it was found that the presence of the substituent at the 3-position influences the nature of the oxidation product. Melatonin and 3-methylindole yielded hydroxylated products, whereas indole and 5-methoxyindole produce chlorinated products.     

Water, proton, and urea transport in toad bladder endosomes that contain the vasopressin-sensitive water channel

Vasopressin (VP) increases the water permeability of the toad urinary bladder epithelium by inducing the cycling of vesicles containing water channels to and from the apical membrane of granular cells. In this study, we have measured several functional characteristics of the endosomal vesicles that participate in this biological response to hormonal stimulation. The water, proton, and urea permeabilities of endosomes labeled in the intact bladder with fluorescent fluid-phase markers were measured. The diameter of isolated endosomes labeled with horse-radish peroxidase was 90-120 nm. Osmotic water permeability (Pf) was measured by a stopped-flow fluorescence quenching assay (Shi, L.-B., and A. S. Verkman. 1989. J. Gen. Physiol. 94:1101-1115). The number of endosomes formed when bladders were labeled in the absence of a transepithelial osmotic gradient increased with serosal [VP] (0-50 mU/ml), and endosome Pf was very high and constant (0.08-0.10 cm/s, 18 degrees C). When bladders were labeled in the presence of serosal-to-mucosal osmotic gradient, the number of functional water channels per endosome decreased (at [VP] = 0.5 mU/ml, Pf = 0.09 cm/s, 0 osmotic gradient; Pf = 0.02 cm/s, 180 mosmol gradient). Passive proton permeability was measured from the rate of pH decrease in voltage-clamped endosomes in response to a 1 pH unit gradient (pHin = 7.5, pHout = 6.5). The proton permeability coefficient (PH) was 0.051 cm/s at 18 degrees C in endosomes containing the VP-sensitive water channel; PH was not different from that measured in vesicles not containing water channels. Measurement of urea transport by the fluorescence quenching assay gave a urea reflection coefficient of 0.97 and a permeability coefficient of less than 10(-6) cm/s. These results demonstrate: (a) VP-induced endosomes from toad urinary bladder have extremely high Pf. (b) In states of submaximal bladder Pf, the density of functional water channels in endosomes in constant in the absence of an osmotic gradient, but decreases in the presence of a serosal-to-mucosal gradient, suggesting that the gradient has a direct effect on the efficiency of packaging of water channels into endosomes. (c) The VP-sensitive water channel does not have a high proton permeability. (d) Endosomes that cycle the water channel do not contain urea transporters. These results establish a labeling procedure in which greater than 85% of labeled vesicles from toad urinary bladder are endosomes that contain the VP-sensitive water channel in a functional form.     

Simultaneous Measurement of Intracellular pH and K+ or NO3- in Barley Root Cells Using Triple-Barreled,Ion-Selective Microelectrodes

The manufacture and use of triple-barreled microelectrodes, which are capable of simultaneous in vivo measurement of intracellular pH and the activities of K+ or NO3- and cell membrane potential (Em), are described. Scanning electron micrographs showed that the three tips were aligned and that the overall tip diameter was approximately 0.8 [mu]m. When filled with 100 mM KCl, all three barrels simultaneously reported identical transmembrane potentials, showing that all three tips were located in the same subcellular compartment. Intracellular estimates of Em in barley (Hordeum vulgare L. cv Klaxon) root epidermal cells obtained with these triple-barreled microelectrodes were indistinguishable from those obtained using single- or double-barreled microelectrodes. Measurements made with triple-barreled K+ and pH-selective microelectrodes in root cells of 7-d-old barley plants grown in a nutrient solution containing 0.5 mM K+ yielded cytosolic and vacuolar populations having mean K+ activity values of 71.3 and 68.7 mM, respectively. The associated mean pH values ([plus or minus]SE) were 7.26 [plus or minus] 0.06 (cytosol) and 5.18 [plus or minus] 0.08 (vacuole). Analysis of whole-tissue digests confirmed the microelectrode measurements. Measurements made using triple-barreled pH- and nitrate-selective microelectrodes confirmed earlier double-barreled measurements of pH and nitrate in barley root epidermal cells growing in 10 mM nitrate.