Solute Accumulation in Tobacco Cells Adapted to NaCl

Cells of Nicotiana tabacum L. var Wisconsin 38 adapted to NaCl (up to 428 millimolar) which have undergone extensive osmotic adjustment accumulated Na⁺ and Cl⁻ as principal solutes for this adjustment. Although the intracellular concentrations of Na⁺ and Cl⁻ correlated well with the level of adaptation, these ions apparently did not contribute to the osmotic adjustment which occurred during a culture growth cycle, because the concentrations of Na⁺ and Cl⁻ did not increase during the period of most active osmotic adjustment. The average intracellular concentrations of soluble sugars and total free amino acids increased as a function of the level of adaptation; however, the levels of these solutes did not approach those observed for Na⁺ and Cl⁻. The concentration of proline was positively correlated with cell osmotic potential, accumulating to an average concentration of 129 millimolar in cells adapted to 428 millimolar NaCl and representing about 80% of the total free amino acid pool as compared to an average of 0.29 millimolar and about 4% of the pool in unadapted cells. These results indicate that although Na⁺ and Cl⁻ are principal components of osmotic adjustment, organic solutes also may make significant contributions.     

Relation between Ion Accumulation of Salt-Sensitive and Isolated Stable Salt-Tolerant Cell Lines of Citrus aurantium

Four selected NaCl-tolerant cell lines of Sour orange (Citrus aurantium) were compared with the nonselected cell line in their growth and internal ion content of Na⁺, K⁺, and Cl⁻ when exposed to increasing NaCl concentrations. No difference was found among the various NaCl-tolerant cell lines in Na⁺ and Cl⁻ uptake, and all these cell lines took up similar or even larger amounts of Na⁺ and Cl⁻ than the NaCl-sensitive cell line. Exposure of cells of NaCl-sensitive and NaCl-tolerant lines to equal external concentrations of NaCl, resulted in a greater loss of K⁺ from the NaCl-sensitive cell line. This observation leads to the conclusion that growth and ability to retain high levels of internal K⁺ are correlated. Exposure of the NaCl-tolerant cell lines to salts other than NaCl resulted in even greater tolerance to Na₂SO₄, but rather poor tolerance to K⁺ introduced as either K₂SO₄ or KCl; the latter has a stronger inhibitory effect. The NaCl-sensitive cell line proved to be more sensitive to replacement of Na⁺ by K⁺. Analyses of internal Na⁺, K⁺, and Cl⁻ concentrations failed to identify any particular internal ion concentration which could serve as a reliable marker for salt tolerance.     

Role of Internal Potassium in Maintaining Growth of Cultured Citrus Cells on Increasing NaCl and CaCl(2) Concentrations

Shamouti orange (Citrus sinensis L. Osbeck) salt-tolerant cells were grown under low water potential conditions induced by polyethylene glycol (PEG), NaCl, and CaCl₂. On the basis of equal osmotic potentials, PEG was the least inhibitory, NaCl next, and CaCl₂ the most inhibitory. The relation between growth capacity and ion content can be summarized as follows. (a) Internal K⁺ concentration was a major factor which changed in the presence of PEG, NaCl, and CaCl₂ and probably played a key role in determining growth capacity. (b) Internal concentrations of Na⁺, Ca²⁺, or Cl⁻ could not be directly correlated with growth. (C) Internal Mg²⁺ concentration could be significant only in the presence of high external Ca²⁺ concentrations. (d) The contribution of nitrate and phosphate to the internal osmoticum was negligible. The ratio of external (Ca²⁺)/(Na⁺)² concentration is crucial for growth. Ratios above 0.5 × 10⁻⁴ per millimolar gave maximal protection from adverse effects of NaCl. Growth capacity was found to be determined by the combination of (Ca²⁺)/(Na⁺)² ratio and the absolute external concentration of NaCl. However, a correlation between internal K⁺ concentration and growth capacity seemed independent of external NaCl concentration.     

Maintenance of low cl concentrations in mesophyll cells of leaf blades of barley seedlings exposed to salt stress

The concentrations of vacuolar Na⁺ and Cl⁻ in the epidermal and mesophyll cells of the leaf blade and sheath of Hordeum vulgare seedlings (cv California Mariout and Clipper) were measured by means of quantitative electron probe x-ray microanalysis. A preferential accumulation of Cl⁻ in vacuoles of epidermal cells in both blade and sheath and a low level in mesophyll cells of the blade were evident in plants grown in full strength Johnson solution. The concentration of Cl⁻ in the mesophyll cells of the blade remained at a low level after exposure to 50 or 100 millimolar NaCl for 1 day or to 50 millimolar for 4 days, while at the same time the concentration of Cl⁻ in the epidermis and mesophyll of the sheath showed a dramatic increase. Clipper generally contained more Cl⁻ in the mesophyll cells of the blade than California Mariout. A greater accumulation of Na⁺ in the mesophyll of the sheath relative to that of the blade was only apparent after treatment with 100 millimolar NaCl for 1 day or 50 millimolar for 4 days. These results confirm the suggestion that sheath tissue is capable of accumulating excess Cl⁻ (and to a lesser extent Na⁺) and suggest that the site of regulation of Cl⁻ concentration in the barley leaf is located in the mesophyll cells of the blade.     

Responses of Atriplex spongiosa and Suaeda monoica to Salinity

The growth and tissue water, K⁺, Na⁺, Cl⁻, proline and glycinebetaine contents of the shoots and roots of two Chenopodiaceae, Atriplex spongiosa and Suaeda monoica have been measured over a range of external NaCl salinities. Both species showed some fresh weight response to low salinity mainly due to increased succulence. S. monoica showed both a greater increase in succulence (at low salinities) and tolerance of high salinities than A. spongiosa. Both species had high affinities for Na⁺ and maintained constant but low shoot K⁺ contents with increasing salinity. These trends were more marked with S. monoica in which Na⁺ stimulated the accumulation of K⁺ in roots. An association between high leaf Na⁺ accumulation, high osmotic pressure, succulence, and a positive growth response at low salinities was noted. Proline accumulation was observed in shoot tissues with suboptimal water contents. High glycinebetaine contents were found in the shoots of both species. These correlated closely with the sap osmotic pressure and it is suggested that glycinebetaine is the major cytoplasmic osmoticum (with K⁺ salts) in these species at high salinities. Na⁺ salts may be preferentially utilized as vacuolar osmotica.     

Abscisic Acid Stimulated Osmotic Adjustment and Its Involvement in Adaptation of Tobacco Cells to NaCl

Osmotic adjustment of cultured tobacco (Nicotiana tabacum L. var Wisconsin 38) cells was stimulated by 10 micromolar (±) abscisic acid (ABA) during adaptation to water deficit imposed by various solutes including NaCl, KCl, K₂SO₄, Na₂SO₄, sucrose, mannitol, or glucose. The maximum difference in cell osmotic potential (Ψπ) caused by ABA treatment during adaptation to 171 millimolar NaCl was about 6 to 7 bar. The cell Ψπ differences elicited by ABA were not due to growth inhibition since ABA stimulated growth of cells in the presence of 171 millimolar NaCl. ABA caused a cell Ψπ difference of about 1 to 2 bar in medium without added NaCl. Intracellular concentrations of Na⁺, K⁺, Cl⁻, free amino acids, or organic acids could not account for the Ψπ differences induced by ABA in NaCl treated cells. However, since growth of NaCl treated cells is more rapid in the presence of ABA than in its absence, greater accumulation of Na⁺, K⁺, and Cl⁻ was necessary for ion pool maintenance. Higher intracellular sucrose and reducing sugar concentrations could account for the majority of the greater osmotic adjustment of ABA treated cells. More rapid accumulation of proline associated with ABA treatment was highly correlated with the effects of ABA on cell Ψπ. These and other data indicate that the role of ABA in accelerating salt adaptation is not mediated by simply stimulating osmotic adjustment.     

Chloride accumulation by mung bean root tips: a low affinity active transport system at the plasmalemma

Net uptake of Cl⁻ into root tips of mung bean (Phaseolus aureus) increases steadily with increasing external concentrations from 1 to 60 mm. Membrane potentials were measured to determine the equilibrium concentration of Cl⁻ in the tissue which could be due to diffusion. This concentration was readily exceeded in both the relatively nonvacuolate tips (0 to 1 mm) and the vacuolate, mature upper sectons (1 to 11 mm) of the roots. The activity coefficient of both cytoplasmic and vacuolar Cl⁻, measured with Cl⁻ sensitive microelectrodes, was approximately the same as that of a pure KCl solution of the same concentration. It is concluded that the “second mechanism” of ion uptake involves a large increase in the rate of active transport at the plasmalemma as the external concentration is increased above 1 mm.     

Solutes contributing to osmotic adjustment in cultured plant cells adapted to water stress

Osmotic adjustment was studied in cultured cells of tomato (Lycopersicon esculentum Mill cv VFNT-Cherry) adapted to different levels of external water potential ranging from −4 bar to −28 bar. The intracellular concentrations of reducing sugars, total free amino acids, proline, malate, citrate, quaternary ammonium compounds, K⁺, NO₃⁻, Na⁺, and Cl⁻ increased with decreasing external water potential. At any given level of adaptation, the maximum contribution to osmotic potential was from reducing sugars followed by potassium ions. The sucrose levels in the cells were 3- to 8-fold lower than reducing sugar levels and did not increase beyond those observed in cells adapted to −16 bar water potential. Concentrations of total free amino acids were 4- to 5-fold higher in adapted cells. Soluble protein levels declined in the adapted cell lines, but the total reduced nitrogen was not significantly different after adaptation. Uptake of nitrogen (as NH₄⁺ or NO₃⁻) from the media was similar for adapted and unadapted cells. Although the level of quaternary ammonium compounds was higher in the nonadapted cells than that of free proline, free proline increased as much as 500-fold compared to only a 2- to 3-fold increase observed for quaternary ammonium compounds. Although osmotic adjustment after adaptation was substantial (up to −36 bar), fresh weight (volume increase) was restricted by as much as 50% in the adapted cells. Altered metabolite partitioning was evidenced by an increase in the soluble sugars and soluble nitrogen in adapted cells which occurred at the expense of incorporation of sugar into cell walls and nitrogen into protein. Data indicate that the relative importance of a given solute to osmotic adjustment may change depending on the level of adaptation.     

Physiological Control of Chloride Transport in Chara corallina: I. EFFECTS OF LOW TEMPERATURE, CELL TURGOR PRESSURE, AND ANIONS

The rate of Cl⁻ transport at the plasma membrane of the freshwater alga Chara corallina is investigated with respect to possible in vivo controls acting in addition to the two well established ones of cytoplasmic Cl⁻ and cytoplasmic pH. In contrast with results from many other plant tissues, halides appear to be the only anions capable of inhibiting Cl⁻ transport, either from the outside or inside surfaces of the plasma membrane. Cell turgor pressure was also investigated. It was found that neither the influx of Cl⁻ nor that of K⁺ or HCO₂⁻ is sensitive to turgor. Internal osmotic pressure is also insensitive to turgor, a situation contrasting with that in closely related brackish water charophytes.     

Quantitative and Physiological Analyses of Chloride Dependence of Growth of Halobacillus halophilus

A quantitative analysis of the Cl⁻ dependence of growth of Halobacillus halophilus was performed. Optimal growth rates were obtained at Cl⁻ concentrations of between 0.5 and 2.0 M, and the final yield was also strictly dependent on the Cl⁻ concentration. Br⁻ but not I⁻, SO₄²⁻, NO₂⁻, SO₂⁻, OCN⁻, SCN⁻, BO₂⁻, or BrO₃⁻ could substitute for Cl⁻. To analyze the function of chloride, chloride concentration was determined. At low external Cl⁻ (Cl_e⁻) concentrations, the growth rate was low and Cl⁻ was excluded from the cytoplasm; increasing the Cl_e⁻ concentration led to an increase in the growth rate and an energy-dependent uptake of Cl⁻, thus decreasing the Cl_e⁻/internal Cl_i⁻ gradient from ≥10 at 0.1 M Cl_e⁻ to a nearly constant value of 2 at Cl_e⁻ concentrations which allowed optimal growth. Two membrane proteins with apparent molecular masses of 31 and 16 kDa which were identified to be specific for Cl⁻-grown cultures are possible candidates for a chloride uptake system.     

Turgor regulation inValonia macrophysa following acute osmotic shock

Summary The marine algaValonia macrophysa an inhabitant of shallow subtropical waters, is subjected to sudden dilutions of external seawater during rain showers. This study describes the mechanisms involved in turgor pressure regulation following acute hyposmotic shock. Turgor regulation is 88% effective and complete within 4 hr following hyposmotic shocks of up to –10 bar. Loss of vacuolar K⁺, Na⁺ and Cl^– accounts for the decrease in vacuolar osmotic pressure associated with turgor regulation. A novel mechanism of turgor regulation is exhibited byValonia macrophysa given hyposmotic shocks greater than about –4 bar. Such an osmotic shock causes cell wall tension to increase above a critical value of about 6×10⁵ dyne/cm, whereupon the protoplasm ruptures and the cell wall stretches irreversibly at a localized site. The protoplasm rupture is suggested by (1) a large abrupt increase in K⁺ efflux (as measured by⁸⁶Rb⁺), (2) a rapid decrease in turgor pressure as measured with a pressure probe, and (3) sudden depolarization of the vacuole potential. Evidence for an increase in cell wall permeability includes efflux from the vacuole of dextran (mol wt 70,000), which normally has a very low cell wall permeability, and scanning electron micrographs which show a trabeculated scar area in the cell wall. This mechanism of turgor regulation is physiologically important because 98% of the cells regained normal growth rate and turgor following acute osmotic shock.     

Stomatal and Nonstomatal Components to Inhibition of Photosynthesis in Leaves of Capsicum annuum during Progressive Exposure to NaCl Salinity

Young bell pepper (Capsicum annuum L.) plants grown in nutrient solution were gradually acclimated to 50, 100, or 150 moles per cubic meter NaCl, and photosynthetic rates of individual attached leaves were measured on several occasions during the salinization period at external CO₂ concentrations ranging from approximately 70 to 1900 micromoles per mole air. Net CO₂ assimilation (A) was plotted against computed leaf internal CO₂ concentration (C_i), and the initial slope of this A-C_i curve was used as a measure of photosynthetic ability. During the 10 to 14 days after salinization began, leaves from plants exposed to 50 moles per cubic meter NaCl showed little change in photosynthetic ability, whereas those treated to 100 or 150 moles per cubic meter NaCl had up to 85% inhibition, with increase in CO₂ compensation point. Leaves appeared healthy, and leaf chlorophyll content showed only a 14% reduction at the highest salinity levels. Partial stomatal closure occurred with salinization, but reductions in photosynthesis were primarily nonstomatal in origin. Photosynthetic ability was inversely related to the concentration of either Na⁺ or Cl⁻ in the leaf laminas sampled at the end of the experimental period. However, the concentration of Cl⁻ expressed on a tissue water basis was greater, exceeding 300 moles per cubic meter, and Cl⁻ was more closely associated (R² = 0.926) with the inhibition of photosynthetic ability. Leaf turgor was not reduced by salinization and leaf osmotic potential decreased to a slightly greater extent than the osmotic potential decreases of the nutrient solutions. Concentration of accumulated Na⁺ and Cl⁻ (on a tissue water basis) accounted quantitatively for maintenance of leaf osmotic balance, assuming that these ions were sequestered in the vacuoles.     

Availability of Chloride Affects the Balance between Potassium Chloride and Potassium Malate in Guard Cells of Vicia faba L

Electron probe microanalysis for K and Cl and enzymic determination of malate were performed on epidermal strips of Vicia faba L. which had been incubated with 0.1 equivalent of K⁺ per liter in the absence or presence of Cl⁻. In the absence of Cl⁻, iminodiacetate, a presumed impermeant zwitterion, served as anion. With no Cl⁻ in the medium, 91% of the K⁺ imported into the guard cells during stomatal opening was neutralized by malate production; import of Cl⁻ (presumably from the rest of the epidermal tissue) contributed 6%. In the presence of Cl⁻, 50% of the necessary negative charges were provided by malate synthesis, 45% by Cl⁻ import. Stomatal opening was not obviously affected by the chloride concentration in the incubation medium, but malate production declined roughly linearly with the logarithm of [Cl⁻] between 10⁻⁵ and 10⁻¹ equivalent per liter.     

Characterization of a proton-translocating ATPase in microsomal vesicles from corn roots

Sealed microsomal vesicles were prepared from corn (Zea mays, Crow Single Cross Hybrid WF9-Mo17) roots by centrifugation of a 10,000 to 80,000g microsomal fraction onto a 10% dextran T-70 cushion. The Mg²⁺-ATPase activity of the sealed vesicles was stimulated by Cl⁻ and NH₄⁺ and by ionophores and protonophores such as 2 micromolar gramicidin or 10 micromolar carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP). The ionophore-stimulated ATPase activity had a broad pH optimum with a maximum at pH 6.5. The ATPase was inhibited by NO₃⁻, was insensitive to K⁺, and was not inhibited by 100 micromolar vanadate or by 1 millimolar azide.

Quenching of quinacrine fluorescence was used to measure ATP-dependent acidification of the intravesicular volume. Quenching required Mg²⁺, was stimulated by Cl⁻, inhibited by NO₃⁻, was insensitive to monovalent cations, was unaffected by 200 micromolar vanadate, and was abolished by 2 micromolar gramicidin or 10 micromolar FCCP. Activity was highly specific for ATP. The ionophore-stimulated ATPase and ATP-dependent fluorescence quench both required a divalent cation (Mg²⁺ ≥ Mn²⁺ > Co²⁺) and were inhibited by high concentrations of Ca²⁺. The similarity of the ionophore-stimulated ATPase and quinacrine quench and the responses of the two to ions suggest that both represent the activity of the same ATP-dependent proton pump. The characteristics of the proton-translocating ATPase differed from those of the mitochondrial F₁F₀-ATPase and from those of the K⁺-stimulated ATPase of corn root plasma membranes, and resembled those of the tonoplast ATPase.

     

Properties of the Isolated Intact Chloroplast at Cytoplasmic K Concentrations : I. Light-Induced Cation Uptake into Intact Chloroplasts is Driven by an Electrical Potential Difference

Photosynthesis, stroma-pH, and internal K⁺ and Cl⁻ concentrations of isolated intact chloroplasts from Spinacia oleracea, as well as ion (K⁺, H⁺, Cl⁻) movements across the envelope, were measured over a wide range of external KCl concentrations (1-100 millimolar).

Isolated intact chloroplasts are a Donnan system which accumulates cations (K⁺ or added Tetraphenylphosphonium⁺) and excludes anions (Cl⁻) at low ionic strength of the medium. The internally negative dark potential becomes still more negative in the light as estimated by Tetraphenylphosphonium⁺ distribution. At 100 millimolar external KCl, potentials both in the light and in the dark and also the light-induced uptake of K⁺ or Na⁺ and the release of protons all become very small. Light-induced K⁺ uptake is not abolished by valinomycin suggesting that the K⁺ uptake is not primarily active. Intact chloroplasts contain higher K⁺ concentrations (112-157 millimolar) than chloroplasts isolated in standard media. Photosynthetic activity of intact chloroplasts is higher at 100 millimolar external KCl than at 5 to 25 millimolar. The pH optimum of CO₂ fixation at high K⁺ concentrations is broadened towards low pH values. This can be correlated with the observation that high external KCl concentrations at a constant pH of the suspending medium produce an increase of stroma-pH both in the light and in the dark. These results demonstrate a requirement of high external concentrations of monovalent cations for CO₂ fixation in intact chloroplasts.

     

Differences between Effects of Undissociated and Anionic 2,4-Dinitrophenol on Permeability of Barley Roots

Effects of 2,4-dinitrophenol (DNP) and several other substituted phenols on permeability of barley roots (Hordeum vulgare var. Trebi) to ions were assayed as a function of pH and phenol concentration. Solutions containing 0.1 micromolar undissociated DNP increase the permeability of barley root cells to small ions such as K⁺, Na⁺, Ca²⁺, and Cl⁻ with no inhibition of respiration. Undissociated forms of the other phenols increase permeability also, but they are less effective than DNP. Only the undissociated DNP is effective. Anionic DNP does not increase permeability or inhibit ion uptake, although it is the major species accumulated by the roots, both at pH 5 and pH 7. At pH 7, in contrast to pH 5, 10 micromolar DNP has no effect on ion permeability of barley roots yet it uncouples oxidative phosphorylation of barley root mitochondria. This indicates that the all too common use of DNP as a test for active transport or involvement of ATP synthesis can be misleading.     

Electrochemical gradients and k and cl fluxes in excised corn roots

The compartmental analysis method was used to estimate the K⁺ and Cl⁻ fluxes for cells of excised roots of Zea mays L. cv. Golden Bantam. When the measured fluxes are compared to those calculated with the Ussing-Teorell flux-ratio equation, an active inward transport of Cl⁻ across the plasmalemma is indicated; the plasmalemma K⁺ fluxes are not far different from those predicted for passive diffusion, although an active inward transport cannot be precluded. Whether fluxes across the tonoplast are active or passive depends upon the vacuolar potential which is unknown. Assuming no electropotential gradient, the tracer flux ratios are fairly close to those predicted for passive movement. However, if the vacuole is positive by about 10 millivolts relative to the cytoplasm, the data suggest active inward transport for K⁺ and outward transport for Cl⁻.     

Ae4 (Slc4a9) Anion Exchanger Drives Cl? Uptake-dependent Fluid Secretion by Mouse Submandibular Gland Acinar Cells

Transcellular Cl⁻ movement across acinar cells is the rate-limiting step for salivary gland fluid secretion. Basolateral Nkcc1 Na⁺-K⁺-2Cl⁻ cotransporters play a critical role in fluid secretion by promoting the intracellular accumulation of Cl⁻ above its equilibrium potential. However, salivation is only partially abolished in the absence of Nkcc1 cotransporter activity, suggesting that another Cl⁻ uptake pathway concentrates Cl⁻ ions in acinar cells. To identify alternative molecular mechanisms, we studied mice lacking Ae2 and Ae4 Cl⁻/HCO₃⁻ exchangers. We found that salivation stimulated by muscarinic and β-adrenergic receptor agonists was normal in the submandibular glands of Ae2^−/− mice. In contrast, saliva secretion was reduced by 35% in Ae4^−/− mice. The decrease in salivation was not related to loss of Na⁺-K⁺-2Cl⁻ cotransporter or Na⁺/H⁺ exchanger activity in Ae4^−/− mice but correlated with reduced Cl⁻ uptake during β-adrenergic receptor activation of cAMP signaling. Direct measurements of Cl⁻/HCO₃⁻ exchanger activity revealed that HCO₃⁻-dependent Cl⁻ uptake was reduced in the acinar cells of Ae2^−/− and Ae4^−/− mice. Moreover, Cl⁻/HCO₃⁻ exchanger activity was nearly abolished in double Ae4/Ae2 knock-out mice, suggesting that most of the Cl⁻/HCO₃⁻ exchanger activity in submandibular acinar cells depends on Ae2 and Ae4 expression. In conclusion, both Ae2 and Ae4 anion exchangers are functionally expressed in submandibular acinar cells; however, only Ae4 expression appears to be important for cAMP-dependent regulation of fluid secretion.     

Microelectrode Measurements on Red Beet Vacuole : Biological Effect of Na OR NO(3) Ions, Diffusing from the Microelectrode

Glass microelectrodes filled with 3 molar KCl are widely used to measure intracellular potentials and it is usual to try to minimize their electrolyte loss. In these experiments we have used the ionic leak of our microelectrodes, filled with various salt solutions, to introduce a given ion into the red beet vacuole. This allowed us to show that NO₃⁻ ions reduce the magnitude of the current spectral density while they do not change the resistance of the tonoplast. This is true when NO₃⁻ is either added to the external medium or used as the microelectrode filling solution. This can be interpreted by a NO₃⁻ effect on the vacuolar side of the tonoplast, resulting in an inhibition of the ion transporting ATPase. Replacing K⁺ by Na⁺ ions in the medium has no effect on tonoplast resistance (R_s). On the contrary, when ions leaking from the microelectrode are H⁺, Li⁺ or K⁺, R_s is close to 4 kilohm square centimeter, whereas R_s is of the order of 30KΩ square centimeter when Na⁺ are the leaking ions. We also found a possible correlation between the presence of a Lorentzian in the current spectral density (cut-off frequency = 2 hertz) and a Cl⁻ efflux from the vacuole. This could be explained by the existence of Cl⁻ channels on the tonoplast.     

Diffusion and Electric Mobility of Ions within Isolated Cuticles of Citrus aurantium: Steady-State and Equilibrium Values

We report a new method for measuring cation and anion permeability across cuticles of sour orange, Citrus aurantium, leaves. The method requires the measurement of two electrical parameters: the diffusion potential arising when the two sides of the cuticle are bathed in unequal concentrations of a Cl⁻ salt; and the electrical conductance of the cuticle measured at a salt concentration equal to the average of that used in the diffusion-potential measurement. The permeabilities of H⁺, Li⁺, Na⁺, K⁺, and Cs⁺ ranged from 2 × 10⁻⁸ to 0.6 × 10⁻⁸ meters per second when cuticles were bathed in 2 moles per cubic meter Cl⁻ salts. The permeability of Cl⁻ was 3 × 10⁻⁹ meters per second. The permeability of Li⁺, Na⁺, and K⁺ was about five times less when measured in 500 moles per cubic meter Cl⁻ salts. We also report an asymmetry in cuticle-conductance values depending on the magnitude and the direction of current flow. The asymmetry disappears at low current-pulse magnitude and increases linearly with the magnitude of the current pulse. This phenomenon is explained in terms of transport-number effects in a bilayer model of the cuticle. Conductance is not augmented by current carried by exchangeable cations in cuticles; conductance is rate limited by the outer waxy layer of the cuticle.