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     

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     

Activation of the Cardiac Ryanodine Receptor by Sulfhydryl Oxidation is Modified by Mg2+ and ATP

The reactive disulfide 4,4′-dithiodipyridine (4,4′DTDP) was added to single cardiac ryanodine receptors (RyRs) in lipid bilayers. The activity of native RyRs, with cytoplasmic (cis) [Ca²⁺] of 10⁻⁷ m (in the absence of Mg²⁺ and ATP), increased within ∼1 min of addition of 1 mm 4,4′-DTDP, and then irreversibly ceased 5 to 6 min after the addition. Channels, inhibited by either 1 mm cis Mg²⁺ (10⁻⁷ m cis Ca²⁺) or by 10 mm cis Mg²⁺ (10⁻³ m cis Ca²⁺), or activated by 4 mm ATP (10⁻⁷ m cis Ca²⁺), also responded to 1 mm cis 4,4′-DTDP with activation and then loss of activity. P _o and mean open time (T _o ) of the maximally activated channels were lower in the presence of Mg²⁺ than in its absence, and the number of openings within the long time constant components of the open time distribution was reduced. In contrast to the reduced activation by 1 mm 4,4′-DTDP in channels inhibited by Mg²⁺, and the previously reported enhanced activation by 4,4′-DTDP in channels activated by Ca²⁺ or caffeine (Eager et al., 1997), the activation produced by 1 mm cis 4,4′-DTDP was the same in the presence and absence of ATP. These results suggest that there is a physical interaction between the ATP binding domain of the cardiac RyR and the SH groups whose oxidation leads to channel activation. Received: 8 September 1997/Revised: 20 January 1998     

A Large-Conductance Chloride Channel in Pigmented Ciliary Epithelial Cells Activated by GTPγS

A large-conductance (or maxi-) chloride channel was identified in bovine pigmented ciliary epithelial (PCE) cells using inside-out excised patch clamp recording. The channel had a mean conductance of 293 pS when excised patches were bathed in symmetrical 130 mm NaCl although the conductance decreased to 209 pS when the solution bathing the cytoplasmic face of the patch contained only 33 mm NaCl. The channel was highly selective for chloride, with a P _Cl/P _Na= 24. A flickery, reversible block was produced by the diuretic stilbene 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), while 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) produced a permanent block. The channel was rarely active in cell-attached patches and usually required several minutes of polarization before activity could be detected in excised patches, a process known as metagenesis. Once activated, the channel was voltage-dependent and was mainly open within the voltage range −30 to +30 mV closing when the membrane was polarized to larger values. GTPγS (100 μm) activated the channel with a latency of 170 sec when applied to the cytoplasmic face of patches. This activation was not reversible upon return to control solution within the duration of the experiment. We assess the available evidence and suggest a role for this channel in volume regulation. Received: 24 June 1996/Revised: 18 February 1997     

Activation of Cl− Currents in Cultured Rat Retinal Pigment Epithelial Cells by Intracellular Applications of Inositol-1,4,5-triphosphate: Differences Between Rats with Retinal Dystrophy (RCS) and Normal Rats

Using the whole-cell configuration of the patch-clamp technique, we studied the conditions necessary for the activation of Cl⁻-currents in retinal pigment epithelial (RPE) cells from rats with retinal dystrophy (RCS) and nondystrophic control rats. In RPE cells from both rat strains, intracellular application of 10 μm inositol-1,4,5-triphosphate (IP3) via the patch pipette led to a sustained activation of voltage-dependent Cl⁻ currents, blockable by 1 mm 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). IP3 activated Cl⁻ currents in the presence of a high concentration of the calcium chelator BAPTA (10 mm) in the pipette solution, but failed to do so when extracellular calcium was removed. Intracellular application of 10⁻⁵ m Ca²⁺ via the patch pipette also led to a transient activation of Cl⁻ currents. When the cells were preincubated in a bath solution containing thapsigargin (1 μm) for 5 min before breaking into the whole-cell configuration, IP3 failed to activate voltage-dependent currents. Thus, IP3 led to release of Ca²⁺ from cytosolic calcium stores. This in turn activated an influx of extracellular calcium into the submembranal space by a mechanism as yet unknown, leading to an activation of calcium-dependent chloride currents. In RPE cells from RCS rats, which show an increased membrane conductance for calcium compared to normal rats, we observed an accelerated speed of Cl⁻-current activation induced by IP3 which could be reduced by nifedipine (1 μm). Thus, the increased membrane conductance to calcium in RPE cells from RCS rats changes the response of the cell to the second messenger IP3. Received: 17 July 1995/Revised: 31 January 1996     

5-Lipoxygenase Metabolites of Arachidonic Acid Regulate Volume Decrease by Mudpuppy Red Blood Cells

We examined whether metabolites of arachidonic acid (AA) regulate K⁺ efflux during regulatory volume decrease (RVD) by mudpuppy red blood cells (RBCs). Volume regulation was inhibited by the phospholipase A₂ antagonists mepacrine (10 μm) and ONO-RS-082 (10 μm); the inhibitory effect of ONO-RS-082 was reversed by gramicidin (5 μm). Eicosatetraynoic acid (ETYA, 100 μm), a general antagonist of AA metabolism, also blocked RVD. In addition, volume regulation was inhibited by the lipoxygenase pathway antagonist nordihydroguaiaretic acid (NDGA, 10 μm), the 5 lipoxygenase antagonists AA-861 (5 μm) and curcumin (20 μm), and by the 5-lipoxygenase activating protein inhibitor L-655,298 (5 μm). Inhibition by all four of these agents was reversed with gramicidin. In contrast, the 12- and 15-lipoxygenase pathway inhibitor ethyl-3,4-dihydroxy-benzylidene-cyanoacetate (EDBCA, 1 μm) and the cytochrome P-450 monooxygenase pathway blocker ketoconazole (20 μm) had no effect. On the other hand, the cyclooxygenase pathway inhibitor aspirin (100 μm) slightly enhanced RVD. Consistent with these findings, a K⁺-selective whole cell conductance responsible for K⁺ efflux during cell swelling was inhibited by ONO-RS-082 (10 μm), NDGA (10 μm), AA-861 (5 μm), curcumin (20 μm), and l-655,298 (5 μm). In contrast, EDBCA (1 μm), ketoconazole (20 μm), and indomethacin (10 μm) did not block this whole cell conductance. These results indicate that a channel mediating K⁺ loss during RVD is regulated by a 5-lipoxygenase metabolite of arachidonic acid. Received: 12 December 1996/Revised: 28 February 1997     

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     

Effects of Chemical Growth Retardants on Growth and Development of Sweetpotato (Ipomoea batatas (L.) Lam.) in Vitro

Plant growth retardants were evaluated for their ability to reduce the growth rate of sweetpotato (Ipomoea batatas (L.) Lam.) in vitro. Nodal sections of cv. Jewel were cultured for 30 days on medium containing NDA, ancymidol, phosfon, TIBA, difenzoquat, chlormequat, ACC, mepiquat chloride, or daminozide at 0, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, or 10⁻⁸ m. Difenzoquat, NDA, phosfon, and TIBA, at 10⁻⁴ m, were lethal to axillary bud explants. A low concentration (10⁻⁸ m) of chlorflurenol or NDA stimulated shoot elongation. The effective concentration range for most growth retardants was 10⁻⁵ to 10⁻⁶ m. Small (2- to 4-mm diameter) storage root-like swellings were observed on roots in cultures containing TIBA or ancymidol. The growth-inhibiting effects of ancymidol and NDA were transitory and did not persist through a 180-day culture period. Shoots cultured on medium containing 10⁻⁵ m phosfon, TIBA, or difenzoquat were significantly shorter than control plants after a 180-day culture period. Culture on medium containing TIBA, NDA, ancymidol, or ACC resulted in abnormal leaf and stem development. Plants derived from nodal explants cultured on medium containing either phosfon or chlormequat were near normal in appearance but with some plants exhibiting interveinal chlorosis and reduced root system development. Received May 9, 1997; accepted August 14, 1997     

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     

Hormonal Effects on Growth and Morphology of Normal and Hairy Roots of Hyoscyamus muticus

Treatment of normal and Agrobacterium rhizogenes-transformed root cultures of Hyoscyamus muticus with three different auxins, indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and naphthaleneacetic acid (NAA), revealed that the response varied considerably among auxins, between transformed and normal roots, and depending on the parameter. In normal roots all three auxins provoked abundant branching, with IBA and NAA being the most effective at 2.5 and 0.5 μm, respectively, whereas IAA was most effective at low concentrations (0.05 and 0.1 μm). In transformed roots exogenously supplied auxins were generally inhibitory or, at best, without effect on growth and branching. Only 0.01 μm IAA significantly enhanced lateral root number, whereas at the higher concentrations IBA, although inhibitory, was the least effective auxin. In both root types IBA had little effect on primary root growth, but normal roots were more sensitive to IAA and NAA. These results suggest a different sensitivity to auxins of normal and transformed roots since there was no significant difference in endogenous free and conjugated IAA content nor in IAA uptake capacity. Ethylene production and biosynthesis were approximately threefold higher in hairy roots, but production could be stimulated up to tenfold that of control levels in normal roots by supplying NAA or 1-aminocyclopropane-1-carboxylic acid (ACC). Treatment with 2.5 μm NAA, but not IAA or IBA, also enhanced ethylene biosynthesis in normal roots but not in transformed ones. ACC and malonyl-1-aminocyclopropane-1-carboxylic acid accumulated to detectable levels only after treatment with an auxin (NAA). Received March 3, 1997; accepted May 28, 1997     

Inhibition by cAMP of Calcium-Activated Chloride Currents in Cultured Sertoli Cells from Immature Testis

We have characterized a Ca²⁺-dependent Cl⁻ current (Cl_Ca) in cultured Sertoli cells from immature rat testis by using the whole cell recording patch-clamp technique. Cells dialyzed with pipette solutions containing 3 mm adenoside-triphosphate (ATP) and 1 μm free Ca²⁺, exhibited outward currents which were inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and anthracene-9-carboxylic acid (9-AC) but insensitive to tetraethylammonium (TEA). Dialysis of cells with pipette solutions containing less than 1 nm free Ca²⁺ strongly reduced the currents indicating that they were Ca²⁺ dependent. With cells dialyzed with Cs⁺ glutamate-rich pipette solutions containing 0.2 mm EGTA, 10 μm ionomycin induced outward currents having properties of Ca²⁺-activated Cl⁻ currents. With ATP-free pipette solution, the magnitude of currents was not modified suggesting the direct control by Ca²⁺. By contrast, addition of 0.1 mm cAMP in the pipette solution or the superfusion of cells by a permeant analogue of cAMP strongly reduced the currents. These results may suggest that Cl_Ca is inhibited by cAMP-dependent protein kinase. Finally, our results do not agree with the model of primary fluid secretion by exocrine cells, but are in agreement with a hyperpolarizing effect of cAMP in primary culture of Sertoli cells and the release of a low Cl⁻ and bicarbonate-rich primary fluid by these cells. Received: 30 November 1998/Revised: 2 March 1999     

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     

Separate Swelling- and Ca2+-activated Anion Currents in Ehrlich Ascites Tumor Cells

A Ca²⁺-activated (I _Cl,Ca) and a swelling-activated anion current (I _Cl,vol) were investigated in Ehrlich ascites tumor cells using the whole cell patch clamp technique. Large, outwardly rectifying currents were activated by an increase in the free intracellular calcium concentration ([Ca²⁺] _i ), or by hypotonic exposure of the cells, respectively. The reversal potential of both currents was dependent on the extracellular Cl⁻ concentration. I _Cl,Ca current density increased with increasing [Ca²⁺] _i , and this current was abolished by lowering [Ca²⁺] _i to <1 nm using 1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA). In contrast, activation of I _Cl,vol did not require an increase in [Ca²⁺] _i . The kinetics of I _Cl,Ca and I _Cl,vol were different: at depolarized potentials, I _Cl,Ca as activated in a [Ca²⁺] _i - and voltage-dependent manner, while at hyperpolarized potentials, the current was deactivated. In contrast, I _Cl,vol exhibited time- and voltage-dependent deactivation at depolarized potentials and reactivation at hyperpolarized potentials. The deactivation of I _Cl,vol was dependent on the extracellular Mg²⁺ concentration. The anion permeability sequence for both currents was I ⁻ > Cl⁻ > gluconate. I _Cl,Ca was inhibited by niflumic acid (100 μm), 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 μm) and 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS, 100 μm), niflumic acid being the most potent inhibitor. In contrast, I _Cl,vol was unaffected by niflumic acid (100 μm), but abolished by tamoxifen (10 μm). Thus, in Ehrlich cells, separate chloride currents, I _Cl,Ca and I _Cl,vol, are activated by an increase in [Ca²⁺] _i and by cell swelling, respectively. Received: 12 November 1997/Revised: 5 February 1998     

Characterization of ATP-Sensitive Potassium Channels Functionally Expressed in Pituitary GH3 Cells

ATP-sensitive K⁺ (K_ATP) channels have been characterized in pituitary GH₃ cells with the aid of the patch-clamp technique. In the cell-attached configuration, the presence of diazoxide (100 μm) revealed the presence of glibenclamide-sensitive K_ATP channel exhibiting a unitary conductance of 74 pS. Metabolic inhibition induced by 2,4-dinitrophenol (1 mm) or sodium cyanide (300 μm) increased K_ATP channel activity, while nicorandil (100 μm) had no effect on it. In the inside-out configuration, Mg-ATP applied intracellularly suppressed the activity of K_ATP channels in a concentration-dependent manner with an IC₅₀ value of 30 μm. The activation of phospholipase A₂ caused by mellitin (1 μm) was found to enhance K_ATP channel activity and further application of aristolochic acid (30 μm) reduced the mellitin-induced increase in channel activity. The challenging of cells with 4,4′-dithiodipyridine (100 μm) also induced K_ATP channel activity. Diazoxide, mellitin and 4,4′-dithiodipyridine activated the K_ATP channels that exhibited similar channel-opening kinetics. In addition, under current-clamp conditions, the application of diazoxide (100 μm) hyperpolarized the membrane potential and reduced the firing rate of spontaneous action potentials. The present study clearly indicates that K_ATP channels similar to those seen in pancreatic β cells are functionally expressed in GH₃ cells. In addition to the presence of Ca²⁺-activated K⁺ channels, K_ATP channels found in these cells could thus play an important role in controlling hormonal release by regulating the membrane potential. Received: 19 June 2000/Revised: 13 September 2000     

Inhibitory Effects of Methyl Jasmonate on the Germination and Ethylene Production in Cocklebur Seeds

Methyl jasmonate (JA-Me) inhibited the germination of cocklebur (Xanthium pennsylvanicum Wallr.) seeds. The inhibition of the germination of cocklebur seeds treated with JA-Me at concentrations less than 300 μm was nullified by ethylene applied exogenously, although the inhibitory effect of 1,000 μm JA-Me was not recovered completely even by high concentrations of ethylene (10,000 μL/liter). JA-Me inhibited ethylene production before seed germination. The level of 1-aminocyclopropane-1-carboxylic acid (ACC) in the cotyledonary tissues treated with JA-Me decreased but not the level of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC). JA-Me inhibited the conversion of ACC to ethylene in the tissues. These results suggested that JA-Me inhibits ethylene production by prevention of ACC oxidation in addition to ACC synthesis. We believe that the inhibition of ethylene production by JA-Me results in the retardation of the germination of cocklebur seeds. Received June 4, 1997; accepted October 23, 1997     

Membrane Potassium Channels and Human Bladder Tumor Cells: II. Growth Properties

These experiments were done to determine the effect of glibenclamide and diazoxide on the growth of human bladder carcinoma (HTB-9) cells in vitro. Cell growth was assayed by cell counts, protein accumulation, and ³H-thymidine uptake. Glibenclamide added at 75 and 150 μm for 48 hr reduced cell proliferation. Dose-inhibition curves showed that glibenclamide added for 48 hr reduced cell growth at concentrations as low as 1 μm (IC₅₀= 73 μm) when growth was assayed in the absence of added serum. This μM-effect on cell growth was in agreement with the dose range in which glibenclamide decreased open probability of membrane K_ATP channels. Addition of glibenclamide for 48 hr also altered the distribution of cells within stages of the cell cycle as determined by flow cytometry using 10⁻⁵ m bromodeoxyuridine. Glibenclamide (100 μm) increased the percentage of cells in G₀/G₁ from 33.6% (vehicle control) to 38.3% (P < 0.05), and it reduced the percentage of cells in S phase from 38.3% to 30.6%. On the other hand, diazoxide, which opens membrane K_ATP channels in HTB-9 cells, stimulated growth measured by protein accumulation, but it did not increase the cell number. We conclude that the sulfonylurea receptor and the corresponding membrane K_ATP channel are involved in mechanisms controlling HTB-9 cell growth. However, K_ATP is not rate-limiting among the signaling mechanisms or molecular switches that regulate the cell cycle. Received: 12 June 1997/Revised: 21 October 1997     

A Possible Role for Jasmonic Acid in Adaptation of Barley Seedlings to Salinity Stress

The changes caused by NaCl salinity and jasmonic acid (JA) treatment (8 days) on growth and photosynthesis of barley plants (Hordeum vulgare L., var. Alfa) have been studied. Gas exchange measurements and analysis of enzyme activities were used to study the reactions of photosynthesis to salinity and JA. Both 100 mm NaCl and 25 μm JA treatment led to a noticeable decrease in both the initial slope of the curves representing net photosynthetic rate vs intercellular CO₂ concentration and the maximal rate of photosynthesis. The calculated values of the intercellular CO₂ concentration, CO₂ compensation point, and maximal carboxylating efficiency of ribulose-1,5-bisphosphate carboxylase support the suggestion that biochemical factors are involved in the response of photosynthesis to JA and salinity stress. The activities of phosphoenolpyruvate carboxylase and carbonic anhydrase increased more than twofold. Pretreatment with JA for 4 days before salinization diminished the inhibitory effect of high salt concentration on the growth and photosynthesis. The results are discussed in terms of a possible role of JA in increasing salinity tolerance of the barley plants. Received September 8, 1997; accepted May 19, 1998     

Activation by Angiotensin II of Ca2+-dependent K+ and Cl− Currents in Zona Fasciculata Cells of Bovine Adrenal Gland

The effects of angiotensin II (100 nm) on the electrical membrane properties of zona fasciculata cells isolated from calf adrenal gland were studied using the whole cell patch recording method. In current-clamp condition, angiotension II induced a biphasic membrane response which began by a transient hyperpolarization followed by a depolarization more positive than the control resting potential. These effects were abolished by Losartan (10⁻⁵ m), an antagonist of angiotensin receptors of type 1. The angiotensin II-induced transient hyperpolarization was characterized in voltage-clamp condition from a holding potential of −10 mV. Using either the perforated or the standard recording method, a transient outward current accompanied by an increase of the membrane conductance was observed in response to the hormonal stimulation. This outward current consisted of an initial fast peak followed by an oscillating or a slowly decaying plateau current. In Cl⁻-free solution, the outward current reversed at −78.5 mV, a value close to E _K. It was blocked by external TEA (20 mm) and by apamin (50 nm). In K⁺-free solution, the transient outward current, sensitive to Cl⁻ channel blocker DPC (400 μm), reversed at −52 mV, a more positive potential than E _Cl. Its magnitude changed in the same direction as the driving force for Cl⁻. The hormone-induced transient outward current was never observed when EGTA (5 mm) was added to the pipette solution. The plateau current was suppressed in nominally Ca²⁺-free solution (47% of cells) and was reversibly blocked by Cd²⁺ (300 μm) but not by nisoldipine (0.5–1 μm) which inhibited voltage-gated Ca²⁺ currents identified in this cell type. The present experiments show that the transient hyperpolarization induced by angiotensin II is due to Ca²⁺-dependent K⁺ and Cl⁻ currents. These two membrane currents are co-activated in response to an internal increase of [Ca²⁺] _i originating from intra- and extracellular stores. Received: 29 May 1997/Revised: 4 November 1997     

Effects of Exogenously Applied Jasmonates on Growth and Intracellular pH in Maize Coleoptile Segments

The effects of jasmonic acid (JA) on elongation growth of coleoptile segments from etiolated maize (Zea mays L.) were investigated in the presence and absence of auxin. When supplied alone, at physiological concentrations (10⁻⁹, 10⁻⁸, and 10⁻⁵ m), JA (or methyl-JA) inhibited growth. JA at a similar range of concentrations also inhibited auxin-induced elongation growth. To determine whether this effect on growth depended on endogenous abscisic acid (ABA), we grew maize coleoptiles in the presence of norflurazon (an inhibitor of carotenoid biosynthesis) that results in reduced endogenous ABA levels. Growth of etiolated coleoptile segments from these plants was inhibited by JA (or methyl-JA) in both the absence and presence of auxin. Previously, we have observed a correlation between elongation growth and cytosolic pH (pH_i), in which auxin lowers pH_i, and growth inhibitors such as ABA raise pH_i. We examined the effect of low concentrations of methyl-JA on pH_i with dual emission dye, carboxy seminaphthorhodafluor-1, and confocal microscopy. To confirm these studies, we also used in vivo ³¹P NMR spectrometry to ascertain the changes in pH_i after addition of jasmonate to maize coleoptiles. Coleoptiles grown in either the absence or presence of norflurazon responded to methyl-JA or JA by increases in pH_i of approximately 0.2 pH unit. This response occurs over a period of 15–20 min and appears to be independent of endogenous ABA. This alkalization induced by JA is likely to form a permissive environment for JA signal transduction pathway(s). Received February 5, 1999; accepted August 25, 1999     

Protein Kinase C and Regulatory Volume Decrease in Mudpuppy Red Blood Cells

This study examined whether protein kinase C (PKC) stimulates K⁺ efflux during regulatory volume decrease (RVD) in Necturus maculosus (mudpuppy) red blood cells (RBCs). The limit of osmotic fragility increased with the general protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 10 μm), but not with the cyclic nucleotide-dependent kinase antagonists N-(2′-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004, 10 μm) and N-2-(methylamino)ethyl-5-isoquinoline-sulfonamide (H-8, 5 μm). Consistent with these results, osmotic fragility also increased with the PKC antagonists bisindolylmaleimide I (GF-109203X or bis I, 100 nm), bisindolylmaleimide II (bis II, 100 nm), and chelerythrine (10 μm). The effect of these three antagonists and H-7 was reversed with gramicidin (5 μm in a choline Ringer), indicating PKC was linked to K⁺ efflux (gramicidin is a cationophore that was used to ensure a high K⁺ permeability). We also measured cell volume recovery from hypotonic shock (0.5× Ringer) with a Coulter counter and estimated cell volume from the hematocrit. The percent RVD compared to control decreased with H-7 (10 μm), sphingosine (100 nm), chelerythrine (10 μm), bis I (100 nm), and bis II (100 nm), but not with HA-1004 (10 μm) nor H-8 (5 μm). Inhibition of RVD by H-7, chelerythrine, bis I, and bis II was reversed with gramicidin (5 μm). Furthermore, using the patch clamp technique, we found H-7 (10 μm) reduced a whole cell conductance that was activated during cell swelling. In addition, a conductance responsible for K⁺ efflux during cell swelling was inhibited by bis I (100 nm) and bis II (100 nm). These results indicate that a conductive pathway mediating K⁺ loss during RVD is regulated, at least in part, by protein kinase C. Received: 20 January 1998/Revised: 2 September 1998