Exogenous Jasmonic and Abscisic Acids Act Differentially in Elongating Tissues from Oat Stem Segments

Segments can be cut from the peducular-1 internode of oat (Avena sativa L.) shoots so as to contain the graviresponsive, auxin-sensitive leaf sheath pulvinus, and the gibberellin-sensitive internodal tissue. These two growth-capable tissues were used to study the effects and interactions of jasmonic acid (JA) and abscisic acid (ABA) in regulating cell elongation. When supplied alone at physiologic concentrations (10⁻⁵, 10⁻⁴ m), JA promoted growth and cell wall synthesis in the internodal tissue, whereas by itself, ABA inhibited internodal elongation and even inhibited JA-promoted growth. When gibberellic acid (GA₃) was used to stimulate internodal elongation, JA and ABA caused similar levels of inhibition and, at certain concentrations, were synergistic. Inhibition by ABA was initiated several hours earlier than inhibition by JA, and only the ABA effect could be partially overcome by 10⁻³ m aminoethoxyvinylglycine. Both JA and ABA inhibited elongation of pulvinar tissue that was induced to grow by gravistimulus or auxin, although here JA was more potent than ABA at equimolar concentrations. When 10⁻⁵ m fusicoccin was used as a general nonphysiologic growth stimulus, JA had no effect on the internode but inhibited the pulvinus, whereas ABA had no effect on the pulvinus but inhibited the internode. These results provide strong physiologic evidence that JA and ABA act by different mechanisms in the regulation of elongation, at least in this representative grass. Received May 28, 1996; accepted November 7, 1996     

Cyanide Inhibition of Chloride Conductance Across Toad Skin

The effect of cyanide (CN⁻) on voltage-activated or cAMP-induced passive chloride conductance (G ^Cl ) was analyzed in isolated toad skin. Comparatively low concentrations of CN⁻ inhibited G ^Cl almost completely and fully reversibly, regardless of whether it was applied from the mucosal or serosal side. The IC₅₀ was 180 ± 12 μm for voltage-activated G ^Cl and 305 ± 30 μm for the cAMP-inducted conductance. At [CN] <100 μm, the initial inhibition frequently declined partly in the continuous presence of CN⁻. Inhibition was independent of the presence of Ca²⁺. Inhibition was stronger at more alkaline pH, which suggests that dissociated CN⁻ is the effective inhibitor. The onset of the inhibition of voltage-activated or cAMP-induced G ^Cl by CN⁻ occurred with half-times of 34 ± 10 sec, whereas reversibility upon washout was twice as fast (18 ± 7 sec). If [CN⁻] <200 μm was applied under inactivating conditions (serosa −30 mV), the reduction of G ^Cl was stronger upon subsequent voltage-activation than under steady-state activated conditions. This effect was essentially complete less than 30 sec after apical addition of CN⁻, but G _t recovered thereafter partially in the continuous presence of CN⁻. Dinitrophenol inhibited G ^Cl similarly, while omission of oxygen did not affect it. These observations, as well as the time course of inhibition and the full reversibility, suggest that interference of CN⁻ with oxidative phosphorylation and subsequent metabolic depletion is not the reason for the inhibition of G ^Cl . We propose that the inhibition is directly on G ^Cl , presumably by competition with Cl⁻ at a rate-limiting site in the pathway. Location and molecular nature of this site remain to be identified. Received: 8 February 1999/Revised: 22 September 1999     

Chloride Currents Activated by Calcitonin and cAMP in Primary Cultures of Rabbit Distal Convoluted Tubule

Chloride (Cl⁻) conductances were studied in primary cultures of the bright part of rabbit distal convoluted tubule (DCTb) by the whole cell patch clamp technique. The bath solution (33°C) contained (in mm): 140 NaCl, 1 CaCl₂, 10 N-2-hydroxy-ethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4 and the pipette solution 140 N-methyl-d-glucamine (NMDG)-Cl, 5 MgATP, 1 ethylene-glycol-bis(b-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 HEPES, pH 7.4. We identified a Cl⁻ current activated by 10⁻⁵ m forskolin, 10⁻³ m 8-bromo adenosine 3′,5′-cyclic monophophosphate (8 Br-cAMP), 10⁻⁶ m phorbol 12-myristate 13-acetate (PMA), 10⁻³ m intracellular adenosine 3′,5′-cyclic monophophosphate (cAMP) and 10⁻⁷ m calcitonin. The current-voltage relationship was linear and the relative ion selectivity was Br⁻ > Cl⁻≫ I⁻ > glutamate. This current was inhibited by 10⁻³ m diphenylamine-2-carboxylate (DPC) and 10⁻⁴ m 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and was insensitive to 10⁻³ m 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). These characteristics are similar to those described for the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ conductance. In a few cases, forskolin and calcitonin induced an outwardly rectifying Cl⁻ current blocked by DIDS. To determine the exact location of the Cl⁻ conductance 6-methoxy-1-(3-sulfonatopropyl) quinolinium (SPQ) fluorescence experiments were carried out. Cultures seeded on collagen-coated permeable filters were loaded overnight with 5 mm SPQ and the emitted fluorescence analyzed by laser-scan cytometry. Cl⁻ removal from the apical solution induced a Cl⁻ efflux which was stimulated by 10⁻⁵ m forskolin, 10⁻⁷ calcitonin and inhibited by 10⁻⁵ m NPPB. In 140 mm NaBr, forskolin stimulated an apical Br⁻ influx through the Cl⁻ pathway. Forskolin and calcitonin had no effect on the basolateral Cl⁻ permeability. Thus in DCTb cultured cells, exposure to calcitonin activates a Cl⁻ conductance in the apical membrane through a cAMP-dependent mechanism. Received: 5 July 1995/Revised: 21 December 1995     

Inhibition of K/HCO(3) cotransport in squid axons by quaternary ammonium ions

Previous squid-axon studies identified a novel K/HCO₃ cotransporter that is insensitive to disulfonic stilbene derivatives. This cotransporter presumably responds to intracellular alkali loads by moving K⁺ and HCO⁻ ₃ out of the cell, tending to lower intracellular pH (pH_i). With an inwardly directed K/HCO₃ gradient, the cotransporter mediates a net uptake of alkali (i.e., K⁺ and HCO⁻ ₃ influx). Here we test the hypothesis that intracellular quaternary ammonium ions (QA⁺) inhibit the inwardly directed cotransporter by interacting at the intracellular K⁺ site. We computed the equivalent HCO⁻ ₃ influx (J _HCO3) mediated by the cotransporter from the rate of pH_i increase, as measured with pH-sensitive microelectrodes. We dialyzed axons to pH_i 8.0, using a dialysis fluid (DF) free of K⁺, Na⁺ and Cl⁻. Our standard artificial seawater (ASW) also lacked Na⁺, K⁺ and Cl⁻. After halting dialysis, we introduced an ASW containing 437 mm K⁺ and 0.5% CO₂/12 mm HCO⁻ ₃, which (i) caused membrane potential to become transiently very positive, and (ii) caused a rapid pH_i decrease, due to CO₂ influx, followed by a slower plateau-phase pH_i increase, due to inward cotransport of K⁺ and HCO⁻ ₃. With no QA⁺ in the DF, J _HCO3 was ∼58 pmole cm⁻² sec⁻¹. With 400 mm tetraethylammonium (TEA⁺) in the DF, J _HCO3 was virtually zero. The apparent K _i for intracellular TEA⁺ was ∼78 mm, more than two orders of magnitude greater than that obtained by others for inhibition of K⁺ channels. Introducing 100 mm inhibitor into the DF reduced J _HCO3 to ∼20 pmole cm⁻² sec⁻¹ for tetramethylammonium (TMA⁺), ∼24 for TEA⁺, ∼10 for tetrapropylammonium (TPA⁺), and virtually zero for tetrabutylammonium (TBA⁺). The apparent K _i value for TBA⁺ is ∼0.86 mm. The most potent inhibitor was phenyl-propyltetraethylammonium (PPTEA⁺), with an apparent K _i of ∼91 μm. Thus, trans-side quaternary ammonium ions inhibit K/HCO₃ influx in the potency sequence PPTEA⁺ > TBA⁺ > TPA⁺ > TEA⁺≅ TMA⁺. The identification of inhibitors of the K/HCO₃ cotransporter, for which no inhibitors previously existed, will facilitate the study of this transporter. Received: 21 November 2000/Revised: 14 May 2001     

An Effect of Ca2+ on the Intrinsic Cl−-conductance of Rat Kidney Cortex Brush Border Membrane Vesicles

Brush-border membrane vesicles (BBMV) were prepared from superficial rat renal cortex by a divalent²⁺-precipitation technique using either CaCl₂ or MgCl₂. The dependence of the initial [¹⁴C]-d-glucose (or [³H]-l-proline) uptake rate and the extent of the overshoot of d-glucose or l-proline uphill accumulation from solutions containing 100 mm Na⁺ salt, was found to be dependent upon the precipitating divalent cation. With Mg²⁺ precipitation the initial uptake and overshoot accumulation of either d-glucose or l-proline were enhanced compared to BBMV prepared by Ca²⁺ precipitation. When the anion composition of the media was varied (uptake in Cl⁻ media in comparison to gluconate⁻-containing media) it was found that the Cl⁻-dependent component of the initial uptake was markedly depressed with Ca²⁺-prepared BBMV (104.99 ± 33.31 vs. 13.83 ± 1.44 pmoles/sec/mg protein for Mg²⁺ and Ca²⁺ prepared vesicles respectively). When Ca²⁺ was loaded into Mg²⁺ prepared BBMV using a freeze-thaw technique, it was found that the magnitude and Cl⁻ enhancement of d-glucose transport was reduced in a dose-dependent manner. Neomycin, an inhibitor of phospholipase C, had no effect on the reduction of d-glucose uptake by Ca²⁺ in Mg²⁺ prepared vesicles. In contrast, phosphatase inhibitors such as vanadate and fluoride were able to partially reverse the Ca²⁺ inhibition of d-glucose uptake and restore the enhancement due to Cl⁻ media. In addition, inhibitors of protein phosphatase 2B, deltamethrin (50 nm) and trifluoperazine (10 μm), caused partial reversal of Ca²⁺-dependent inhibition of d-glucose uptake. Direct measurement of changes in the bi-ionic (Cl⁻ vs. gluconate⁻) transmembrane electrical potential differences using the cyanine dye, 3,3′-dipropylthiodicarbocyanine iodide DiSC₃-(5) confirmed that Cl⁻ conductance was reduced in Ca²⁺-prepared vesicles. We conclude that a Cl⁻ conductance coexists with Na⁺ cotransport in rat renal BBMV and this may be subject to negative regulation by Ca²⁺ via stimulation of protein phosphatase (PP2B). Received: 14 December 1994/Revised: 27 November 1995     

Kinetic Analysis of the Inhibitory Effect of Glibenclamide on KATP Channels of Mammalian Skeletal Muscle

We investigated the block of K_ATP channels by glibenclamide in inside-out membrane patches of rat flexor digitorum brevis muscle. (1) We found that glibenclamide inhibited K_ATP channels with an apparent K _i of 63 nm and a Hill coefficient of 0.85. The inhibition of K_ATP channels by glibenclamide was unaffected by internal Mg²⁺. (2) Glibenclamide altered all kinetic parameters measured; mean open time and burst length were reduced, whereas mean closed time was increased. (3) By making the assumption that binding of glibenclamide to the sulphonylurea receptor (SUR) leads to channel closure, we have used the relation between mean open time, glibenclamide concentration and K _D to estimate binding and unbinding rate constants. We found an apparent rate constant for glibenclamide binding of 9.9 × 10⁷ m ⁻¹ sec⁻¹ and an unbinding rate of 6.26 sec⁻¹. (4) Glibenclamide is a lipophilic molecule and is likely to act on sulfonylurea receptors from within the hydrophobic phase of the cell membrane. The glibenclamide concentration within this phase will be greater than that in the aqueous solution and we have taken this into account to estimate a true binding rate constant of 1.66 × 10⁶ m ⁻¹ sec⁻¹. Received: 7 July 1996/Revised: 4 October 1996     

Regulation of aromatic amino acid biosynthesis in the ribulose monophosphate cycle methylotroph Nocardia sp. 239

The regulation of aromatic amino acid biosynthesis in Nocardia sp. 239 was studied. In cell-free extracts 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase activity was inhibited in a cumulative manner by tryptophan, phenylalanine and tyrosine. Chorismate mutase was inhibited by both phenylalanine and tyrosine, whereas prephenate dehydratase was very sensitive to inhibition by phenylalanine. Tyrosine was a strong activator of the latter enzyme, whereas anthranilate synthase was inhibited effectively by tryptophan. No clear repression of the synthesis of these enzymes was observed during growth of the organism in the presence of the aromatic amino acids. It is therefore concluded that in Nocardia sp. 239 synthesis of these amino acids is mainly regulated by feedback inhibition. The molecular organization and kinetic properties of DAHP synthase were studied in more detail following its purification. The molecular weight of the native enzyme and its single subunit species were estimated to be 168,000 and 41,000, respectively, suggesting that the enzyme is a tetramer. Apparent K _m values for phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) were 45 and 370 M, respectively. Tryptophan, phenylalanine and tyrosine inhibited DAHP synthase in a competitive manner with respect to E4P, with apparent K _i values of 3, 160 and 180 M, respectively. In addition, tryptophan and E4P (apparent K _i values of 11 and 530 M, respectively) were found to exert an uncompetitive and competitive inhibition, respectively, towards PEP.Abbreviations DAHP 3-deoxy-D-arabino-heptulosonate 7-phosphate - E4P erythrose-4-phosphate - PEP phosphoenolpyruvate - RuMP ribulose monophosphate - HPLC high performance liquid chromatography - FPLC fast protein liquid chromatography - SDS sodium dodecyl sulphate     

Physiological and Biochemical Role of Brassinosteroids and Their Structure-Activity Relationship in the Green Alga Chlorella vulgaris Beijerinck (Chlorophyceae)

This paper studies the influence of the 7-oxalactone type of brassinosteroids (BRs) and 6-ketone upon the biological activity of the alga Chlorella vulgaris (Chlorophyceae). The results of the study indicate significant differences in the growth and metabolism of C. vulgaris cells caused by the different chemical structures of the BRs used. The most significant differences in the stimulation of the growth of the biomass and metabolites contained in it were caused by structural differences in the B ring of BRs. It was found that in C. vulgaris 7-oxalactone type of BRs [brassinolide (BL) and its derivatives] are more active than 6-ketone type of BRs [castasterone (CS) and its derivatives]. It was found that BRs used within the range of concentration of 10⁻¹² to 10⁻⁸ m stimulate two- to threefold the growth and division of C. vulgaris cells. The most stimulating influence upon the number of the algal cells and the phosphorus, chlorophyll, and monosaccharides contained in the alga, as well as the intensity of the photosynthesis, and sugar and glycolate excretion was demonstrated by BL at a concentration 10⁻⁸ m in the 36th h of cultivation. HomoCS was characterized by the lowest biological activity. In turn, after the 48th h an inhibition of the rate of growth and development of the alga takes place. In the range from 10⁻⁷ to 10⁻⁶ m the inhibition of growth and development of the alga was manifested by BRs. During the further toxic activity of BRs the cells of C. vulgaris undergo complete degradation. In turn, in concentrations lower than 10⁻¹² m, BRs do not exert any biologically significant influence upon C. vulgaris cells. On the basis of the study, the biological activity of BRs was arranged in the following order: BL > 24-epiBL > homoBL > CS > 24-epiCS > homoCS. Received July 21, 1997; accepted April 7, 1998     

Involvement of Protein Tyrosine Kinase in the InsP3-Induced Activation of Ca2+-Dependent Cl− Currents in Cultured Cells of the Rat Retinal Pigment Epithelium

This combined study of patch-clamp and intracellular Ca²⁺ ([Ca²⁺] _i ) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca²⁺-dependent Cl⁻ channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 μm) led to an increase in [Ca²⁺] _i and activation of Cl⁻ currents. In contrast, intracellular application of Ca²⁺ (10 μm) only induced transient activation of Cl⁻ currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca²⁺ and to the blocker of I _CRAC, La³⁺ (10 μm), despite the fact that both maneuvers led to a decline in [Ca²⁺] _i . The InsP3-induced rise in Cl⁻ conductance could be prevented either by thapsigargin-induced (1 μm) depletion of intracellular Ca²⁺ stores or by removal of Ca²⁺ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca²⁺-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 μm) had no effect. Inhibition of Ca²⁺/calmodulin kinase (KN-63, KN-92; 5 μm) reduced Cl⁻-conductance in 50% of the cells investigated without affecting [Ca²⁺] _i . Inhibition of protein tyrosine kinase (50 μm tyrphostin 51, 5 μm genistein, 5 μm lavendustin) reduced an increase in [Ca²⁺] _i and Cl⁻ conductance. In summary, elevation of [Ca] _i by InsP3 leads to activation of Cl⁻ channels involving cytosolic Ca²⁺ stores and Ca²⁺ influx from extracellular space. Tyrosine kinases are essential for the Ca²⁺-independent maintenance of this conductance. Received: 15 October 1998/Revised: 3 March 1999     

Large Conductance Ca2+-activated K+ Channels in the Soma of Rat Motoneurones

Properties of large conductance Ca²⁺-activated K⁺ channels were studied in the soma of motoneurones visually identified in thin slices of neonatal rat spinal cord. The channels had a conductance of 82 ± 5 pS in external Ringer solution (5.6 mm K⁺ _o //155 mm K⁺ _i ) and 231 ± 4 pS in external high-K _o solution (155 mm K⁺ _o //155 mm K⁺ _i ). The channels were activated by depolarization and by an increase in internal Ca²⁺ concentration. Potentials of half-maximum channel activation (E₅₀) were −13, −34, −64 and −85 mV in the presence of 10⁻⁶, 10⁻⁵, 10⁻⁴ and 10⁻³ m internal Ca²⁺, respectively. Using an internal solution containing 10⁻⁴ m Ca²⁺, averaged K_Ca currents showed fast activation within 2–3 msec after a voltage step to +50 mV. Averaged K_Ca currents did not inactivate during 400 msec voltage pulses. External TEA reduced the apparent single-channel amplitude with a 50% blocking concentration (IC₅₀) of 0.17 ± 0.02 mm. K_Ca channels were completely suppressed by externally applied 100 mm charybdotoxin. It is concluded that K_Ca channels activated by Ca²⁺ entry during the action potential play an important role in the excitability of motoneurones. Received: 7 November 1996/Revised: 29 October 1997     

The Swelling-Activated Anion Conductance in the Mouse Renal Inner Medullary Collecting Duct Cell Line mIMCD-K2

Swelling-activated Cl⁻ currents (I _Cl,swell ) have been characterized in a mouse renal inner medullary collecting duct cell line (mIMCD-K2). Currents activated by exposing the cells to hypotonicity exhibited characteristic outward rectification and time- and voltage-dependent inactivation at positive potentials and showed an anion selectivity of I⁻ > Br⁻ > Cl⁻ > Asp⁻. NPPB (100 μm) inhibited the current in a voltage independent manner, as did exposure to 10 μm tamoxifen and 500 μm niflumic acid (NFA). In contrast, DIDS (100 μm) blocked the current with a characteristic voltage dependency. These characteristics of I _Cl,swell in mIMCD-K2 cells are essentially identical to those of heterologously expressed cardiac CLC-3. A defining feature of CLC-3 is that activation of PKC by PDBu inhibits the conductance. In mIMCD-K2 cells preincubation with PDBu (100 nm) prevented the activation of I _Cl,swell by hypotonicity. However, PDBu inhibition of I _Cl,swell was reversed after PDBu withdrawal, but this was refractory to subsequent PDBu inhibition. Activation of either the cystic fibrosis transmembrane conductance regulator (CFTR) or Ca²⁺ activated Cl⁻ conductance (CaCC), which are coexpressed in mIMCD-K2 cells prior to PDBu treatment, abolished the PDBu inhibition of I _Cl,swell . Control of I _Cl,swell by PKC therefore depends on the physiological status of the cell. In intact mIMCD-K2 layers in Ussing chambers, forskolin stimulation of an inward short-circuit current (due to transepithelial Cl⁻ secretion via apical CFTR) was inhibited by cell swelling upon hypotonic exposure at the basolateral surface. Activation of I _Cl,swell is therefore capable of regulating transepithelial Cl⁻ secretion and suggests that I _Cl,swell is located at the basolateral membrane. PDBu exposure prior to or during hypotonic challenge was ineffective in reversing the swelling-activated inhibition of Cl⁻ secretion, but tamoxifen (100 μm) abolished the hypotonic inhibition of forskolin-stimulated short-circuit current (I _sc ). RT-PCR analysis confirmed expression of mRNA for members of the CLC family, including both CLC-2 and 3, in the mIMCD-K2 cell line. Received: 24 February 2000/Revised: 26 May 2000     

In Vitro Culture of Rudimentary Embryos of Ilex paraguariensis: Responses to Exogenous Cytokinins

The effects of benzyladenine (BAP), kinetin (KIN), zeatin (ZEA), isopentenyladenine (2iP), and thidiazuron (TDZ) were studied on in vitro growth of rudimentary embryos of Ilex paraguariensis St. Hil. Heart stage zygotic embryos were removed from seeds of immature, light green fruits and cultured aseptically on quarter-strength Murashige and Skoog medium containing 3% sucrose, 0.65% agar, and supplemented with or without three concentrations of BAP, KIN, ZEA, 2iP, or TDZ. Cultures were incubated in darkness at 27 ± 2°C. Media containing 4.4 × 10⁻⁶ m BAP, 4.6 × 10⁻⁶ m KIN, or 4.9 × 10⁻⁶ m 2iP were totally ineffective in inducing embryo growth after culture for 28 days. However, lower concentrations of these compounds (4.4 × 10⁻⁸ m BAP, 4.6 × 10⁻⁸ m KIN, 4.5 × 10⁻⁸ m ZEA, or 4.9 × 10⁻⁸ m 2iP) promoted embryo growth. TDZ at 9.9 × 10⁻⁹ m, 9.9 × 10⁻⁸ m, or 9.9 × 10⁻⁷ m induced embryo growth at similar rates. The maximum percentage of embryos converted to seedlings was achieved when the medium was supplemented with 4.5 × 10⁻⁷ m ZEA. Received August 1, 1997; accepted February 19, 1998     

Transcellular Chloride Pathways in Ambystoma Proximal Tubule

The transport mechanisms of Ambystoma proximal tubule that mediate transcellular Cl⁻ absorption linked to Na⁺ were investigated in isolated perfused tubules using Cl⁻-selective and voltage-recording microelectrodes. In control solutions intracellular activity of Cl⁻ (a _i ^Cl ) is 11.3 ± 0.5 mm, the basolateral (V ₁ ), apical (V ₂ ), and transepithelial (V ₃ ) potential differences are −68 ± 1.2 mV, +62 ± 1.2 mV and −6.4 ± 0.3 mV, respectively. When Na⁺ absorption is decreased by removal of organic substrates from the lumen, a _i ^Cl falls by 1.3 ± 0.3 mm and V ₂ hyperpolarizes by +11.4 ± 1.7 mV. Subsequent removal of Na⁺ from the lumen causes a _i ^Cl to fall further by 2.3 ± 0.4 mm and V ₂ to hyperpolarize further by +15.3 ± 2.4 mV. The contribution of transporters and channels to the observed changes of a _i ^Cl was examined using ion substitutions and inhibitors. Apical Na/Cl or Na/K/2Cl symport is excluded because bumetanide, furosemide or hydrochlorothiazide have no effect on a _i ^Cl . The effects of luminal HCO⁻ ₃ removal and/or of disulfonic stilbenes argue against the presence of apical Cl-base exchange such as Cl-HCO₃ or Cl-OH. The effects of basolateral HCO⁻ ₃ removal, of basolateral Na⁺ removal and/or of disulfonic stilbenes are compatible with presence of basolateral Na-independent Cl-base exchange and Na-driven Cl-HCO₃ exchange. Several lines of evidence favor conductive Cl⁻ transport across both the apical and basolateral membrane. Addition of the chloride-channel blocker diphenylamine-2-carboxylate to the lumen or bath, increases the a _i ^Cl by 2.4 ± 0.6 mm or 2.9 ± 1.0 mm respectively. Moreover, following inhibition by DIDS of all anion exchangers in HCO⁻ ₃-free Ringer, the equilibrium potential for Cl⁻ does not differ from the membrane potential V ₂ . Finally, the logarithmic changes in a _i ^Cl in various experimental conditions correlate well with the simultaneous changes in either basolateral or apical membrane potential. These findings strongly support the presence of Cl⁻ channels at the apical and basolateral cell membranes of the proximal tubule. Received: 14 November 1997/Revised: 6 July 1998     

Phenylalanine hydroxylase and isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase in relationship to the phylogenetic position of Pseudomonas acidovorans (Ps. sp. ATCC 11299a)

The evolution of aromatic amino acid biosynthesis and its regulation is under study in a large assemblage of prokaryotes (Superfamily A) whose phylogenetic arrangement has been constructed on the criterion of oligonucleotide cataloging. One section of this Superfamily consists of a well defined (rRNA homology) cluster denoted as Group III pseudomonads. Pseudomonas acidovorans ATCC 11299a, a Group III member, was chosen for indepth studies of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase, the initial regulatory enzyme of aromatic biosynthesis. This strain is of particular interest for evolutionary studies of aromatic metabolism because it possesses phenylalanine hydroxylase, an enzyme whose physiological role and distribution among prokaryotes is largely unknown. Although P. acidovorans ATCC 11299a has been of uncertain identity, we now establish it unambiguously as a species of acidovorans by virtue of its 87% DNA homology with P. acidovorans ATCC 15668 (type strain). This result conformed with enzyme patterning studies which placed ATCC 11299a into pseudomonad Group IIIa, a subgroup containing the acidovorans species. Crude extracts of Group III pseudomonads had previously been shown to share, as a common group characteristic, sensitivity of DAHP synthase to feedback inhibition by either l-tyrosine or l-phenylalanine. Detailed studies with partially purified preparations from strain ATCC 11299a revealed the presence of two distinct regulatory isozymes, DAHP synthase-phe and DAHP synthase-tyr. DAHP synthase-tyr is tightly controlled by l-tyrosine with 50% inhibition of activity being achieved at 4.0 M effector. DAHP synthase-phe is inhibited 50% by 40 M l-phenylalanine and exhibits dramatic changes in levels of activity, as well as chromatographic elution patterns, in response to dithiothreitol. This two-isozyme pattern of DAHP synthase has not been described previously, although it may prove to be widespread.Abbreviations DAHP 3-deoxy-d-arabino-heptulosonate 7-phosphate - E4P d-erythrose-4-phosphate - PEP phosphoenolpyruvate - DTT dithiothreitol - BSA fraction V bovine serum albumin     

A Novel,Volume-correlated Cl− Conductance in Marginal Cells Dissociated from the Stria Vascularis of Gerbils

Using the whole-cell patch-clamp technique, we examined Cl⁻-selective currents manifested by strial marginal cells isolated from the inner ear of gerbils. A large Cl⁻-selective conductance of ∼18 nS/pF was found from nonswollen cells in isotonic buffer containing 150 mm Cl⁻. Under a quasi-symmetrical Cl⁻ condition, the `instantaneous' current-voltage relation was close to linear, while the current-voltage relation obtained at the end of command pulses of duration 400 msec showed weak outward rectification. The permeability sequence for anionic currents was as SCN⁻ > Br⁻≅ Cl⁻ > F⁻ > NO⁻ ₃≅ I⁻ > gluconate⁻, corresponding to Eisenmann's sequence V. When whole-cell voltage clamped in isotonic bathing solutions, the cells exhibited volume changes that were accounted for by the Cl⁻ currents driven by the imposed electrochemical potential gradients. The volume change was elicited by lowered extracellular Cl⁻ concentration, anion substitution and altered holding potentials. The Cl⁻ conductance varied in parallel with cell volume when challenged by bath anisotonicity. The whole-cell Cl⁻ current was only partially blocked by both 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 0.5 mm) and diphenylamine-2-carboxylic acid (DPC, 1.0 mm), but 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid (SITS, 0.5 mm) was without effect. The properties of the present whole-cell Cl⁻ current resembled those of the single Cl⁻ channel previously found in the basolateral membrane of the marginal cell (Takeuchi et al., Hearing Res. 83:89–100, 1995), suggesting that the volume-correlated Cl⁻ conductance could be ascribed predominantly to the basolateral membrane. This Cl⁻ conductance may function not only in cell volume regulation but also for the transport of Cl⁻ and the setting of membrane potential in marginal cells under physiological conditions. Received: 15 August 1995/Revised: 3 November 1995     

HCO3 − Transport in a Mathematical Model of the Pancreatic Ductal Epithelium

We have used computer modeling to investigate how pancreatic duct cells can secrete a fluid containing near isotonic (∼140 mm) NaHCO₃. Experimental data suggest that NaHCO₃ secretion occurs in three steps: (i) accumulation of HCO⁻ ₃ across the basolateral membrane of the duct cell by Na(HCO₃) _n cotransporters, Na⁺/H⁺ exchangers and proton pumps; (ii) secretion of HCO⁻ ₃ across the luminal membrane on Cl⁻/HCO⁻ ₃ antiporters operating in parallel with Cl⁻ channels; and (iii) diffusion of Na⁺ through the paracellular pathway. Programming the currently available experimental data into our computer model shows that this mechanism for HCO⁻ ₃ secretion is deficient in one important respect. While it can produce a relatively large volume of a HCO⁻ ₃-rich fluid, it can only raise the luminal HCO⁻ ₃ concentration up to about 70 mm. To achieve secretion of 140 mm NaHCO₃ by the model it is necessary to: (i) reduce the conductive Cl⁻ permeability and increase the conductive HCO⁻ ₃ permeability of the luminal membrane of the duct cell, and (ii) reduce the activity of the luminal Cl⁻/HCO⁻ ₃ antiporters. Under these conditions most of the HCO⁻ ₃ is secreted via a conductive pathway. Based on our data, we propose that HCO⁻ ₃ secretion occurs mainly by the antiporter in duct segments near the acini (luminal HCO⁻ ₃ concentration up to ∼70 mm), but mainly via channels further down the ductal tree (raising luminal HCO⁻ ₃ to ∼140 mm). Received: 15 November 1999/Revised: 29 March 2000     

Vacuolar Chloride Transport in Mesembryanthemum crystallinum L. Measured Using the Fluorescent Dye Lucigenin

To study vacuolar chloride (Cl⁻) transport in the halophilic plant Mesembryanthemum crystallinum L., Cl⁻ uptake into isolated tonoplast vesicles was measured using the Cl⁻-sensitive fluorescent dye lucigenin (N,N′-dimethyl-9,9′-bisacridinium dinitrate). Lucigenin was used at excitation and emission wavelengths of 433 nm and 506 nm, respectively, and showed a high sensitivity towards Cl⁻, with a Stern-Volmer constant of 173 m ⁻¹ in standard assay buffer. While lucigenin fluorescence was strongly quenched by all halides, it was only weakly quenched, if at all, by other anions. However, the fluorescence intensity and Cl⁻-sensitivity of lucigenin was shown to be strongly affected by alkaline pH and was dependent on the conjugate base used as the buffering ion. Chloride transport into tonoplast vesicles of M. crystallinum loaded with 10 mm lucigenin showed saturation-type kinetics with an apparent K _m of 17.2 mm and a V _max of 4.8 mm min⁻¹. Vacuolar Cl⁻ transport was not affected by sulfate, malate, or nitrate. In the presence of 250 μm p-chloromercuribenzene sulfonate, a known anion-transport inhibitor, vacuolar Cl⁻ transport was actually significantly increased by 24%. To determine absolute fluxes of Cl⁻ using this method, the average surface to volume ratio of the tonoplast vesicles was measured by electron microscopy to be 1.13 × 10⁷ m⁻¹. After correcting for a 4.4-fold lower apparent Stern-Volmer constant for intravesicular lucigenin, a maximum rate of Cl⁻ transport of 31 nmol m⁻² sec⁻¹ was calculated, in good agreement with values obtained for the plant vacuolar membrane using other techniques. Received: 18 February 2000/Revised: 30 June 2000     

ADH Action on Whole-Cell Currents by Cytosolic Ca2+-dependent Pathways in Aldosterone-treated A6 Cells

We studied the characteristics of the basal and antidiuretic hormone (arginine vasotocin, AVT)-activated whole cell currents of an aldosterone-treated distal nephron cell line (A6) at two different cytosolic Ca²⁺ concentrations ([Ca²⁺] _c , 2 and 30 nm). A6 cells were cultured on a permeable support filter for 10 ∼ 14 days in media with supplemental aldosterone (1 μm). At 30 nm [Ca²⁺] _c , basal conductances mainly consisted of Cl⁻ conductances, which were sensitive to 5-nitro-2-(3-phenylpropylamino)-benzoate. Reduction of [Ca²⁺] _c to 2 nm abolished the basal Cl⁻ conductance. AVT evoked Cl⁻ conductances at 2 as well as 30 nm [Ca²⁺] _c . In addition to Cl⁻ conductances, AVT induced benzamil-insensitive nonselective cation (NSC) conductances. This action on NSC conductances was observed at 30 nm [Ca²⁺] _c but not at 2 nm [Ca²⁺] _c . Thus, cytosolic Ca²⁺ regulates NSC and Cl⁻ conductances in a distal nephron cell line (A6) in response to AVT. Keeping [Ca²⁺] _c at an adequate level seems likely to be an important requirement for AVT regulation of ion conductances in aldosterone-treated A6 cells. Received: 6 May 1996/Revised: 28 June 1996     

High-Affinity NO− 3-H+ Cotransport in the Fungus Neurospora: Induction and Control by pH and Membrane Voltage

High-affinity nitrate transport was examined in intact hyphae of Neurospora crassa using electrophysiological recordings to characterize the response of the plasma membrane to NO⁻ ₃ challenge and to quantify transport activity. The NO⁻ ₃-associated membrane current was determined using a three electrode voltage clamp to bring membrane voltage under experimental control and to compensate for current dissipation along the longitudinal cell axis. Nitrate transport was evident in hyphae transferred to NO⁻ ₃-free, N-limited medium for 15 hr, and in hyphae grown in the absence of a nitrogen source after a single 2-min exposure to 100 μm NO⁻ ₃. In the latter, induction showed a latency of 40–80 min and rose in scalar fashion with full transport activity measurable approx. 100 min after first exposure to NO⁻ ₃; it was marked by the appearance of a pronounced sensitivity of membrane voltage to extracellular NO⁻ ₃ additions which, after induction, resulted in reversible membrane depolarizations of (+)54–85 mV in the presence of 50 μm NO⁻ ₃; and it was suppressed when NH₄ ⁺ was present during the first, inductive exposure to NO⁻ ₃. Voltage clamp measurements carried out immediately before and following NO⁻ ₃ additions showed that the NO⁻ ₃-evoked depolarizations were the consequence of an inward-directed current that appeared in parallel with the depolarizations across the entire range of accessible voltages (−400 to +100 mV). Measurements of NO⁻ ₃ uptake using NO⁻ ₃-selective macroelectrodes indicated a charge stoichiometry for NO⁻ ₃ transport of 1(+):1(NO⁻ ₃) with common K _m and J _max values around 25 μm and 75 pmol NO⁻ ₃ cm⁻²sec⁻¹, respectively, and combined measurements of pH _o and [NO⁻ ₃] _o showed a net uptake of approx. 1 H⁺ with each NO⁻ ₃ anion. Analysis of the NO⁻ ₃ current demonstrated a pronounced voltage sensitivity within the normal physiological range between −300 and −100 mV as well as interactions between the kinetic parameters of membrane voltage, pH _o and [NO⁻ ₃] _o . Increasing the bathing pH from 5.5 to 8.0 reduced the current and the associated membrane depolarizations 2- to 4-fold. At a constant pH _o of 6.1, driving the membrane voltage from −350 to −150 mV resulted in an approx. 3-fold reduction in the maximum current and a 5-fold rise in the apparent affinity for NO⁻ ₃. By contrast, the same depolarization effected an approx. 20% fall in the K _m for transport as a function in [H⁺] _o . These, and additional results are consistent with a charge-coupling stoichiometry of 2(H⁺) per NO⁻ ₃ anion transported across the membrane, and implicate a carrier cycle in which NO⁻ ₃ binding is kinetically adjacent to the rate-limiting step of membrane charge transit. The data concur with previous studies demonstrating a pronounced voltage-dependence to high-affinity NO⁻ ₃ transport system in Arabidopsis, and underline the importance of voltage as a kinetic factor controlling NO⁻ ₃ transport; finally, they distinguish metabolite repression of NO⁻ ₃ transport induction from its sensitivity to metabolic blockade and competition with the uptake of other substrates that draw on membrane voltage as a kinetic substrate. Received: 17 March 1997/Revised: 20 June 1997