Internode Length in Pisum. A New Allele at the le Locus

In Pisum sativum L. a third, more severe, allele at the internodelength locus le is identified and named le^d. Plants homozygousfor le^d possess shorter internodes and appear relatively lessresponsive to GA₂₀ than comparable le (dwarf) plants. Gene le^dmay act by reducing the 3ß-hydroxylation of GA₂₀ tothe highly active GA₁ more effectively than does gene le. Theresults indicate that le is a leaky mutant and therefore thatendogenous GA₁ influences internode elongation in dwarf (le)plants. Pisum sativum, peas, internode length, genetics, gibberellin, dwarf elongation     

Internode length in pisum: do the internode length genes effect growth in dark-grown plants?

Internode length in light-grown peas (Pisum sativum L.) is controlled by the interaction of genes occupying at least five major loci, Le, La, Cry, Na, and Lm. The present work shows that the genes at all of the loci examined (Le, Cry, and Na) also exert an effect on internode length in plants grown in complete darkness. Preliminary results using pure lines were verified using either segregating progenies or near isogenic lines. The major cause of the differences was due to a change in the number of cells per internode rather than to an alteration of the cell length. Since the genes occupying at least two of these loci, Le and Na, have been shown to be directly involved with gibberellin metabolism, it appears that gibberellins are not only essential for elongation in the dark but are limiting for elongation in the nana (extremely short, na), dwarf (Na le), and tall (Na Le) phenotypes. These results are supported by the large inhibitory effects of AMO 1618 treatments on stem elongation in dwarf and tall lines grown in the dark and the fact that applied gibberellic acid could overcome this inhibition and greatly promote elongation in a gibberellin-deficient na line. It is clear that the internode length genes, and in particular the alleles at the Le locus, are not acting by simply controlling the sensitivity of the plant to light.     

Gibberellins in Light-Grown Shoots of Pisum sativum L. and the Influence of Reproductive Development

The presence of GA₉, GA₁₉ and GA₂₀ was demonstrated by gas chromatography/massspectrometry (GC/MS) and the presence of GA₄₄ strongly indicatedby GC/MS in selected ion monitoring mode (GC/SIM) in extractsof shoots of light-grown tall peas (Pisum sativum L.). Usingthe rice seedling bioassay with cv. Tan-ginbozu, the levelsof gibberellins in pea shoots were monitored from early shootgrowth through to apical senescence in a tall pea line. Levelsof activity corresponding to GA₂₀, GA₁₉ and GA₄₄ remained relativelystable in the shoot despite reproductive development and apicalsenescence. The level of GA₁-like activity increased to a maximumwhen the leaves had between 7 and 9 leaves expanded and decreasedonly with apical senescence. The na gene which blocks the productionof biologically active gibberellins in shoots but not in developingseed, was also operative in pod walls, with na pods containinglittle or no significant gibberellin-like activity when comparedto na pods at contact. This occurred despite the presence ofrelatively high levels of gibberellins in developing seed atthe same time. The results suggest that there is little or nosignificant leakage of biologically active gibberellins fromdeveloping seed to pods or shoots. Extracts of pods of tallpeas with Na contained low levels of gibberellin-like activitybut like developing seed, contained little or no significantGA₁-like activity despite the presence of significant GA₁-likeactivity in shoot extracts of tall peas. (Received March 11, 1986; Accepted May 27, 1986)     

Internode Length in Pisum: Variation in Response to a Daylength Extension with Incandescent Light

Lines representing a range of internode length and floweringgenotypes in Pisum sativum L. were grown in 8 h of daylightfollowed by either 16 h of darkness or incandescent light. Thestem elongation response index (RI = length in 24 h ÷length in 8 h) was least in the very short internode nana types,which are grossly deficient in gibberellins (GAs), and the verylong internode slender types, which behave as if saturated withGAs. The common tall (genotype Le) and dwarf (le) types (lepartially blocks conversion of GA₂₀ to the active form, GA₁)were all markedly responsive but the peak RI (based on the mostresponsive internode) was less in tall lines (1.79 to 2.78)than in dwarf lines (2.32 to 5.01) and the peak RI tended tooccur about three to four internodes earlier in tall than indwarf lines. The cry⁸ mutation reduced the RI. (Duplicate lengthloci La and Cry are probably concerned with GA reception.) Amongle dwarf lines, genotype La cry⁸, was generally less responsivethan La Cry, La cry^c and la Cry. Data from crosses showed thaton either an le La or le la background cry⁸ segregates had alower RI than cry⁸ segregates. On an le la background, cry⁸plants were shorter than cry^c plants, cry⁸ was partially dominantto cry⁸ and segregation was clear only in long days. On an lela background, cry^c plants were shorter than cry^c plants, cry⁸was partially dominant to cry⁸ and segregation was clear inlong or short days. The very high peak RI (5.0) of the microcryptodwarfline, L57, appeared to result, in part, from a marked foreshorteningof internodes 4 to 10 in the 8 h regime. In the 24 h regimeL57 (lm) had a fairly similar growth pattern to normal (Lm)cryptodwarf types. The peak RI tended to occur at a lower internode in early thanlate flowering lines, especially among dwarf types, and genotypeswith a day neutral flowering habit (genotype sn or dne) wereless responsive than their photoperiodic counterparts (Sn Dne). White fluorescent light, given as a daylength extension, wasmuch less effective than incandescent light at stimulating stemelongation suggesting control through the phytochrome equilibrium(P_tr/P_total). Pisum sativum, garden pea, daylength extension, flowering, genotype, gibberellin, hormone receptor, incandescent light, internode length, phytochrome, stem elongation     

Endogenous Gibberellins in the Vegetative Shoots of Tall and Dwarf Cultivars of Phaseolus vulgaris L.

A series of 13-hydroxygibberellins, gibberellin A₁ (GA₁), GA₁₉,GA₂₀, GA₄₄ and GA₅₃, were identified by GC/MS (full scan) fromvegetative shoots of tall (cv. Kentucky Wonder) and dwarf (cv.Masterpiece) Phaseolus vulgaris L. It is suggested that GA₁is active per se in the control of shoot elongation of P. vulgarisL., and that dwarfism in Masterpiece is not due to shortageof the active GA, but to its low ability to respond to the bioactiveGA. (Received August 29, 1988; Accepted November 21, 1988)     

Gibberellin concentration and transport in genetic lines of pea : effects of grafting

Effects of the Na and Le loci on gibberellin (GA) content and transport in pea (Pisum sativum L.) shoots were studied. GA₁, GA₈, GA₁₇, GA₁₉, GA₂₀, GA₂₉, GA₄₄, GA₈ catabolite, and GA₂₉ catabolite were identified by full-scan gas chromatography-mass spectrometry in extracts of expanding and fully expanded tissues of line C79-338 (Na Le). Quantification of GAs by gas chromatography-single-ion monitoring using deuterated internal standards in lines differing at the Na and Le alleles showed that na reduced the contents of GA₁₉, GA₂₀, and GA₂₉ on average to <3% and of GA₁ and GA₈ to <30% of those in corresponding Na lines. In expanding tissues from Na le lines, GA₁ and GA₈ concentrations were reduced to approximately 10 and 2%, respectively, and GA₂₉ content increased 2- to 3-fold compared with those in Na Le plants. There was a close correlation between stem length and the concentrations of GA₁ or GA₈ in shoot apices in all six genotypes investigated. In na/Na grafts, internode length and GA₁ concentration of nana scions were normalized, the GA₂₀ content increased slightly, but GA₁₉ levels were unaffected. Movement of labeled GAs applied to leaves on Na rootstocks indicated that GA₁₉ was transported poorly to apices of na scions compared with GA₂₀ and GA₁. Our evidence suggests that GA₂₀ is the major transported GA in peas.     

Quantitative Analysis of Endogenous Gibberellins in Normal and Dwarf Cultivars of Rice

Endogenous levels of gibberellins in shoots and ears of twodwarf rice (Oryza sativa L.) cultivars, Tan-ginbozu (dx mutant)and Waito-C (dy mutant), were analyzed and compared with thoseof normal rice cultivar, Nihonbare. The endogenous levels of13-hydroxylated gibberellins in Tan-ginbozu were much lowerthan those in Nihonbare. In Waito-C, the levels of GA₁₉ andGA₂₀ in the shoots were higher but that of GA₁ was lower thanthe levels of these gibberellins in Nihonbare. These resultssupport the hypothesis that the dy gene controls the 3ß-hydroxylationof GA₂₀ to GA₁ while the dx gene controls a much earlier stepin the gibberellin biosynthesis. Our results indicate that GA₁is the active gibberellin that regulates the vegetative growthof rice. The endogenous levels of GA₄ in the ears of the twodwarf cultivars of rice were higher than the level of GA₄ inthe ears of the normal cultivar, Nihonbare suggesting that thebiosynthesis of gibberellin is not blocked in the anthers ofthe dwarf rice. (Received April 27, 1989; Accepted July 12, 1989)     

Effects of Interactions between Low-temperature Treatments, Gibberellin (GA3) and Photoperiod on Flowering and Stem Height of Spring Rape (Brassica napus var. annua)

Exogenous gibberellin A₃(GA₃) reduced the number of leaf nodesat flowering and time to flowering and increased the stem heightat flowering in three genotypes of spring rape (Brassica napusvar.annua L.). The responses to GA₃were similar to those forlong days (LD) and low-temperature treatments, suggesting thatthe effect of photoperiod and the vernalization response areprobably mediated through gibberellins. The response to exogenousGA₃was greatest in non-cold-treated plants in short days (SD)suggesting that endogenous GAs are limiting in these conditions.CCC, an inhibitor of gibberellin biosynthesis, caused a smallincrease in the number of leaf nodes at flowering and time toflowering and a small decrease in the stem height at flowering,but unexpectedly, its effect was hardly influenced by the applicationof exogenous GA₃. Genotypes that showed the clearest responsesto the treatments with regard to the number of leaf nodes atflowering and time to flowering did not show the clearest responseswith regard to the stem height at flowering; the pattern ofresponses of the number of leaf nodes at flowering and timeto flowering was distinct from that of stem height at flowering.This indicates that flower formation and stem elongation areseparable developmental processes which may be controlled bydifferent endogenous gibberellins, different levels of a specificendogenous gibberellin, or different responses to gibberellin.Copyright 1999 Annals of Botany Company Brassica napus var. annua, gibberellin, photoperiod, spring rape, vernalization.     

Studies of Growth and Flowering in Picea sitchensis (Bong.) Carr. 1. Effects of Growth Regulator Applications to Mature Scions on Seedling Rootstocks

Effects of growth regulator applications on the flowering of5-years-grafted mature scions of Picea sitchensis (Bong.) Carr.were assessed. Growth regulators were applied to the bud surfacein droplets of ethanol during July, August and September ina polythene house or glasshouse. A mixture of gibberellins A₄and A₇ applied alone, and in combination with gibberellins A₃and A₅, significantly increased numbers by up to seven timesfor male and by up to eight times for female strobili, gibberellinA, gave relatively the strongest response, and gibberellin A₄was inactive. Phosphon D and abscisic acid each reversed thepromotion of flowering by gibberellins, whilst kinetin and N,N-diphenylureahad no effect. The number of female strobili was negativelycorrelated with vegetative shoot length in the year after treatment. Under field conditions hormones were applied in July and Augustunder flaps of bark on the branches of 10-years-grafted maturescions. Gibberellin applications caused a 5-fold increase inflowering and N⁶-benzyladenine further increased the response.Naphth-lyl-acetic acid reduced female and increased male flowering.Bark removal near the base of the branch further enhanced hormone-inducedstrobilus production. The usefulness of these findings for thebreeding of Picea sitchensis is discussed.     

Potent block of inactivation-deficient Na+ channels by n-3 polyunsaturated fatty acids

A voltage-gated, small, persistent Na⁺ current (I_Na) has been shown in mammalian cardiomyocytes. Hypoxia potentiates the persistent I_Na that may cause arrhythmias. In the present study, we investigated the effects of n-3 polyunsaturated fatty acids (PUFAs) on I_Na in HEK-293t cells transfected with an inactivation-deficient mutant (L409C/A410W) of the -subunit (hH1) of human cardiac Na⁺ channels (hNav1.5) plus ₁-subunits. Extracellular application of 5 µM eicosapentaenoic acid (EPA; C20:5n-3) significantly inhibited I_Na. The late portion of I_Na (I_{Na late}, measured near the end of each pulse) was almost completely suppressed. I_Na returned to the pretreated level after washout of EPA. The inhibitory effect of EPA on I_Na was concentration dependent, with IC₅₀ values of 4.0 ± 0.4 µM for I_Na peak (I_{Na peak}) and 0.9 ± 0.1 µM for I_{Na late}. EPA shifted the steady-state inactivation of I_{Na peak} by –19 mV in the hyperpolarizing direction. EPA accelerated the process of resting inactivation of the mutant channel and delayed the recovery of the mutated Na⁺ channel from resting inactivation. Other polyunsaturated fatty acids, docosahexaenoic acid, linolenic acid, arachidonic acid, and linoleic acid, all at 5 µM concentration, also significantly inhibited I_Na. In contrast, the monounsaturated fatty acid oleic acid or the saturated fatty acids stearic acid and palmitic acid at 5 µM concentration had no effect on I_Na. Our data demonstrate that the double mutations at the 409 and 410 sites in the D1–S6 region of hH1 induce inactivation-deficient I_Na and that n-3 PUFAs inhibit mutant I_Na. human cardiac sodium channel     

Intracellular pH shifts in cultured kidney (A6) cells: effects on apical Na+ transport

We report, for the epithelialNa⁺ channel (ENaC) in A6 cells,the modulation by cell pH (pH_c)of the transepithelial Na⁺ current(I_Na), thecurrent through the individual Na⁺channel (i), the openNa⁺ channel density(N_o), and thekinetic parameters of the relationship betweenI_Na and theapical Na⁺ concentration. Thei andN_o were evaluatedfrom the Lorentzian I_Na noise inducedby the apical Na⁺ channel blocker6-chloro-3,5-diaminopyrazine-2-carboxamide.pH_c shifts were induced, understrict and volume-controlled experimental conditions, byapical/basolateral NH₄Cl pulses orbasolateral arrest of theNa⁺/H⁺exchanger (Na⁺ removal; block byethylisopropylamiloride) and were measured with the pH-sensitive probe2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Thechanges in pH_c were positivelycorrelated to changes inI_Na and theapically dominated transepithelial conductance. The sole pH_c-sensitive parameter underlyingI_Na wasN_o. Only thesaturation value of theI_Na kinetics wassubject to changes in pH_c.pH_c-dependent changes inN_o may be causedby influencingP_o, the ENaC openprobability, or/and the total channel number,N_T = N_o/P_o.

     

Inheritance of a Mutation which Suppresses Flowering in Red Clover

A spontaneous mutation isolated from stocks of red clover (cultivarS123) prevents flower initiation unless plants are suppliedexogenously with gibberellin. Mutant plants are also more uprightand densely tillering in their growth habit. Inheritance ofthe non-flowering character was analysed in a series of crossesbetween wild-type S123 and mutant plants. Hybridity followingintercrossing was confirmed using electrophoretic variants ofcytoplasmic phosphoglucose isomerase coded by a- and b-allelesof the nuclear gene Pgi-2. All F₁ plants flowered normally andwere heterozygous at the Pgi-2 locus. However, F₂ segregationsdid not provide the expected ratios, with flowering plants exceedingpredicted levels. One back-cross involving an F₁ plant and themutant parent gave flowering:non flowering and ab:aa Pgi-2 ratiosof 2:1 rather than the expected 1:1. The results are consistentwith the existence of a zygotic lethal factor, originally presentin heterozygous (non-lethal) form in the mutant (non-flowering)parent and tightly linked to the mutated gene. Segregants whichwere non-flowering always displayed the characteristic mutantgrowth form and seeds borne on these plants were lighter incolour than those borne on normal plants. Thus, there existsin red clover a gene designated ‘dig’ (developmentinfluencing gibberellin) which has several pleiotropic effectsincluding suppressing the initiation of flowering in normallyflorally-inductive environments. There are at least two allelicforms of the gene, F (flowering) and f (non-flowering).     

Effects of Gibberellin on Shoot Development in the dgt Mutant of Tomato

The morphological effects of gibberellin A₃ (GA₃) on the dgtmutant of tomato were investigated. The mutant effectively showedthe normal range of responses, including a promotion of stemlength due to an increased number of longer internodes, a dramaticincrease in apical dominance, and effects on leaf shape andcolour. In the case of stem elongation, the quantitative responseof the mutant was greater than normal. The morphological abnormalitiescharacteristic of the dgt mutant, such as horizontal growth,a thin stem and hyponastic leaves, were not normalized by GA₃. It is concluded that the demonstrated lack of response to auxinof the dgt mutant does not impair its gibberellin responses. Tomato, gibberellin, auxin, mutant, shoot development     

Growth Pattern of Tall and Short Lines of Rice and their Response to Gibberellin

The growth pattern of two types of dwarfness in rice was studied.The difference in height of the tall and short Peta plants wasapparent even at the seedling stage. In the dwarf and in thenormal Century Patna 231 the difference in height was apparentonly after panicle initiation. The four lines had the same numberof elongated internodes, with the taller lines having longerinternodes. The application of gibberellin resulted in increased plant height,longer internodes, blades and leaf sheaths, and decreased tillernumber. Century Patna 231-dwarf showed the least, and shortPeta the greatest, response to gibberellin application.     

Effect of Sodium on Phosphate Uptake in Unicellular and Filamentous Cyanobacteria

The effect of Na⁺ on phosphate uptake was studied in four strainsof cyanobacteria: Synechococcus PCC 7942, Gloeothece PCC 6501,Phormidium sp. and Chlorogloeopsis PCC 6912. Phosphate uptakewas stimulated by Na⁺ in all cases. Li⁺ and K⁺ acted as partialanalogues for Na⁺. Half-saturation [K_1/2(Na⁺)] of phosphateuptake was reached with Na⁺ concentrations ranging from 317µM in Chlorogloeopsis to 659 µM in Phormidium. Theconcentration of phosphate required to reach half-saturationof phosphate uptake [K_1/2(P_i)]was not changed by the presenceof Na⁺. (Received April 11, 1994; Accepted July 5, 1994)     

Ionic Relations of Garden Orache, A triplex hortensis L.: Growth and Ion Distribution at Moderate Salinity and the Function of Bladder Hairs

The growth of garden orache, A triplex hortensis was studiedunder conditions of mild NaCl or Na₂SO₄ salinity. Growth, drymatter production and leaf size were substantially stimulatedat 10 mM and 50 mM Na⁺ salts. Increased growth, however, appearedto be due to a K⁺-sparing effect of Na⁺ rather than to salinityper se. The distribution of K⁺ and Na⁺ in the plant revealeda remarkable preference for K+ in the roots and the hypocotyl.In the shoot the K/Na ratio decreased strongly with leaf age.However, the inverse changes in K⁺ and Na⁺ content with leafage were dependent on the presence of bladder hairs, which removedalmost all of the Na⁺ from the young leaf lamina. Measurementsof net fluxes of K⁺ and Na⁺ into roots and shoots of growingAtriplex plants showed a higher K/Na selectivity of the netion flux to the root compared to the shoot. With increasingsalinity the selectivity ratio S_{K, Na}^* of net ion fluxes tothe roots and to the shoots was increased. The data suggestthat recirculation of K⁺ from leaves to roots is an importantlink in establishing the K/Na selectivity in A. hortensis plants.The importance of K⁺ recirculation and phloem transport forsalt tolerance is discussed. Key words: Atriplex hortensis, Salinity, Potassium, Sodium, K⁺ retranslocation, Bladder hairs, Growth stimulation     

Effect of the Rht3 dwarfing gene on dynamics of cell extension in wheat leaves, and its modification by gibberellic acid and paclobutrazol

The second leaf of wheat was used as a model system to examinethe effects of the Rht3 dwarfing gene on leaf growth. Comparedto the rht3 wild type, the Rht3allele decreased final leaf length,surface area and dry mass by reducing the maximum growth rates,but without affecting growth duration. Gibberellic acid (GA₃)increased final leaf length and maximum growth rate in the rht3wild type, but was without effect on the Rht3 mutant, whichis generally regarded as being non-responsive to gibberellin(GA). Paclobutrazol, an inhibitor of GA biosynthesis, decreasedfinal leaf length and maximum growth rate in the rht3 wild typeto values similar to those in the untreated Rht3 mutant. NeitherGA₃ nor paclobutrazol affected the duration of leaf growth.The decrease in leaf length was produced by reduction of celllength rather than cell number. The maximum relative elementalgrowth rate (REGR) for cell extension was essentially the samein all treatments, as was the time between the cells leavingthe meristem and achieving maximum extension rate. The differencesbetween the genotypes and treatments were all almost entirelydue to differences in the time taken from the attainment ofmaximum REGR of cell extension to the cessation of extension.This was reflected in the length of the extension zone, whichwas approximately 6–8 per cent of final leaf length. Theeffects of the Rht3 allele, GA₃ and paclobutrazol all appearto be on the processes which promote the cessation of cell elongation. Key words: Cell extension, gibberellin, leaf growth, Rht3 gene, Triticum, wheat     

Internode length inPisum: biochemical expression of thele andna mutations in the slender phenotype

Thele andna mutations in pea block GA biosynthesis and normally cause a marked reduction in internode length. However, neither of these genes influences the growth of plants carrying thecry ^s la gene combination. Plants of this genotype have long, thin internodes, pale green foliage, and abnormal flower and fruit development, collectively referred to as the slender phenotype. [¹³C,³H]Gibberellin A₂₀ is metabolized to GA₁, GA₈, and GA₂₉ in slender lines carrying the geneLe but only to GA₂₉ and GA₂₉-catabolite inle lines. Examination of¹²C:¹³C isotopic ratios showed that metabolites were strongly diluted by endogenous [¹²C]GAs inNa lines. However, little if any significant dilution was observed in a line homozygous for thena gene. These results confirm that thele andna mutations are fully expressed at the biochemical level in slender phenotypes of peas and concur with previous reports that internode elongation is entirely independent of GA levels incry ^s la (slender) plants.     

Molecular determinants of voltage-gated sodium channel regulation by the Nedd4/Nedd4-like proteins

The voltage-gated Na⁺ channels (Na_v) form a family composed of 10 genes. The COOH termini of Na_v contain a cluster of amino acids that are nearly identical among 7 of the 10 members. This COOH-terminal sequence, PPSYDSV, is a PY motif known to bind to WW domains of E3 protein-ubiquitin ligases of the Nedd4 family. We recently reported that cardiac Na_v1.5 is regulated by Nedd4-2. In this study, we further investigated the molecular determinants of regulation of Na_v proteins. When expressed in HEK-293 cells and studied using whole cell voltage clamping, the neuronal Na_v1.2 and Na_v1.3 were also downregulated by Nedd4-2. Pull-down experiments using fusion proteins bearing the PY motif of Na_v1.2, Na_v1.3, and Na_v1.5 indicated that mouse brain Nedd4-2 binds to the Na_v PY motif. Using intrinsic tryptophan fluorescence imaging of WW domains, we found that Na_v1.5 PY motif binds preferentially to the fourth WW domain of Nedd4-2 with a K_d of 55 µM. We tested the binding properties and the ability to ubiquitinate and downregulate Na_v1.5 of three Nedd4-like E3s: Nedd4-1, Nedd4-2, and WWP2. Despite the fact that along with Nedd4-2, Nedd4-1 and WWP2 bind to Na_v1.5 PY motif, only Nedd4-2 robustly ubiquitinated and downregulated Na_v1.5. Interestingly, coexpression of WWP2 competed with the effect of Nedd4-2. Finally, using brefeldin A, we found that Nedd4-2 accelerated internalization of Na_v1.5 stably expressed in HEK-293 cells. This study shows that Nedd4-dependent ubiquitination of Na_v channels may represent a general mechanism regulating the excitability of neurons and myocytes via modulation of channel density at the plasma membrane. ubiquitin; Nedd4-2; PY motif; Na_v1.5; human ether-à-go-go-related gene     

Hormonal regulation of ENaCs: insulin and aldosterone

Although a variety of hormones and other agents modulate renalNa⁺ transport acting by way of theepithelial Na⁺ channel (ENaC), themode(s), pathways, and their interrelationships in regulation of thechannel remain largely unknown. It is likely that several hormones maybe present concurrently in vivo, and it is, therefore, important tounderstand potential interactions among the various regulatory factorsas they interact with the Na⁺transport pathway to effect modulation ofNa⁺ reabsorption in distal tubulesand other native tissues. This study represents specifically adetermination of the interaction between two hormones, namely,aldosterone and insulin, which stimulate Na⁺ transport by entirelydifferent mechanisms. We have used a noninvasive pulse protocol ofblocker-induced noise analysis to determine changes in single-channelcurrent (i_Na),channel open probability (P_o), andfunctional channel density(N_T) ofamiloride-sensitive ENaCs at various time points following treatmentwith insulin for 3 h of unstimulated control and aldosterone-pretreatedA6 epithelia. Independent of threefold differences of baseline values of transport caused by aldosterone, 20 nM insulin increased by threefold and within 10-30 min the density of the pool of apical membrane ENaCs(N_T) involvedin transport. The very early (10 min) increases of channel density wereaccompanied by relatively small decreases ofi_Na(10-20%) and decreases ofP_o (28%) in the aldosterone-pretreated tissues but not the control unstimulated tissues. The early changes ofi_Na,P_o, andN_T weretransient, returning very slowly over 3 h toward their respectivecontrol values at the time of addition of insulin. We conclude thataldosterone and insulin act independently to stimulate apicalNa⁺ entry into the cells of A6epithelia by increase of channel density.