Transfers between biological and environmental systems

A newM _γ categorical representation of biological systems is developed which is an extension of previously reported work (Leguizamón, 1975a, b; 1976). In this representation, each material physical nature going to a component or produced by it, is given by a set whose cardinality corresponds to the quantity of the material physical nature. Biological and environmental diagrams, respectively represented byM _γ andE categories, are used in order to obtain a theory of transfers between both systems. From the mathematical representation of this theory, the transfers are classified as: material-energetic, exclusively energetic and theoretical material-theoretical energetic. These concepts are incorporated into a diagram, and later, a set of axioms is given, in order to have the previous developments as a new categoryT. This paper was made possible by a Fellowship from the Consejo Nacional de Investigationes Científicas y Técnicas of the República Argentina.     

A note on abstract relational biologies

It is shown that the class of abstract block diagrams of (M, ℜ)-systems which can be constructed out of the objects and mappings of a particular subcategoryG ₀ of the categoryG of all sets depends heavily on the structure ofG ₀, and in particular on the number of sets of mappingsH(A, B) which are empty inG ₀. In the context ofG ₀-systems, there-fore, each particular categoryG ₀ gives rise to a different “abstract biology” in the sense of Rashevsky. A number of theorems illustrating the relation between the structure of a categoryG ₀ and the embeddability of an arbitrary mapping αεG ₀ into an (M, ℜ)-system are proved, and their biological implication is discussed. This research was supported by the United States Air Force through the Air Force Office of Scientific Reserch of the Air Research and Development Command, under Contract No. AF 49(638)-917.     

Study of the oxygen transfer in a disposable flexible bioreactor with surface aeration in vibrated medium

The oxygen mass transfer is a critical design parameter for most bioreactors. It can be described and analyzed by means of the volumetric mass transfer coefficient K _L a. This coefficient is affected by many factors such as geometrical and operational characteristics of the vessels, type, media composition, rheology and microorganism’s morphology and concentration. In this study, we aim to develop and characterize a new culture system based on the surface aeration of a flexible, single-used bioreactor fixed on a vibrating table. In this context, the K _L a was evaluated using a large domain of operating variables such as vibration frequency of the table, overpressure inside the pouch and viscosity of the liquid. A novel method for K _L a determination based on the equilibrium state between oxygen uptake rate and oxygen transfer rate of the system at given conditions was also developed using resting cells of baker’s fresh yeast with a measured oxygen uptake rate of 21 mg g⁻¹ h⁻¹ (at 30°C). The effect of the vibration frequency on the oxygen transfer performance was studied for frequencies ranging from 15 to 30 Hz, and a maximal K _L a of 80 h⁻¹ was recorded at 30 Hz. A rheological study of the medium added with carboxymethylcellulose at different concentrations and the effect of the liquid viscosity on K _L a were determined. Finally, the mixing time of the system was also measured using the pH method.     

Kinetics of lipase-catalyzed hydrolysis of palm oil in lecithin/izooctane reversed micelles

Candida rugosa lipase has been used to investigate the hydrolysis of palm oil in a lecithin/isooctane reversed micellar system. The reaction obeys Michaelis-Menten kinetics for the initial conditions. Kinetic parameters such as maximum rate and Michaelis constant (K _m) were determined for lipase-catalyzed hydrolysis in n-hexane and isooctane. According to the K _m values, the enzyme affinity towards the substrate was increased in isooctane. The maximum degree of hydrolysis was generally decreased as the initial substrate concentration was increased. This may suggest that the hydrolysis in lecithin reversed micelles should be regarded as a one-substrate first-order reversible reaction. It is shown in this study that the proposed one-substrate first-order kinetic model can serve for the precise prediction of the degree of hydrolysis for a known reaction time or vice versa, when the initial substrate concentration is less than 0.325 mol/dm³. A disagreement with this model was found when the initial substrate concentration was higher than approximately 0.3 mol/dm³. This may be due to the effects of the products on lipase activity or even to the conversion of the reversed micellar system to other systems. Received: 16 May 1997 / Received revision: 22 October 1997 / Accepted: 24 October 1997     

The Use of the Free Metal – Temperature ‘Phase Diagrams’ for Studies of Single Site Metal Binding Proteins

Typical physico-chemical studies of metal binding proteins are usually aimed at determination of the metal binding constant K for a native protein (K _n), while the significance of the K value for the thermally denatured protein (K _u) is usually underestimated. Meanwhile, metal binding induced shift of thermal denaturation transition of a single site metal binding protein is defined by K _n to K _u ratio, implying that knowledge of both K values is required for full characterization of the system. In the present work, the most universal approach to the studies of single site metal binding proteins, namely construction of a protein “phase diagram” in coordinates of free metal ion concentration – temperature, is considered in detail. The detailed algorithm of construction of the phase diagrams along with underlying mathematic procedures developed here may be of use for studies of other simple protein-target type systems, where target represents low molecular weight ligand. Analysis of the simplest protein-ligand system reveals that thermodynamic properties of apo-protein dictate the maximal possible increase of its affinity to any simple ligand upon thermal denaturation of the protein. Experimental and general problems coupled with the use of the phase diagrams are discussed.     

Effect of Auxiliary Substances on Complexation Efficiency and Intrinsic Dissolution Rate of Gemfibrozil–β-CD Complexes

The studies reported in this work are aimed to elucidate the ternary inclusion complex formation of gemfibrozil (GFZ), a poorly water-soluble drug, with β-cyclodextrin (β-CD) with the aid of auxiliary substances like different grades of povidone(s) (viz. PVP K-29/32, PVP K-40, Plasdone S-630, and Polyplasdone XL), organic base (viz. triethanolamine), and metal ion (viz. MgCl₂·6H₂O), by investigating their interactions in solution and solid state. Phase solubility studies were carried out to evaluate the solubilizing power of β-cyclodextrin, in association with various auxiliary substances, to determine the apparent stability constant (K _C) and complexation efficiency (CE) of complexes. Improvement in K _C values for ternary complexes clearly proves the benefit of the addition of auxiliary substances to promote CE. Of all the approaches used, the use of polymer Plasdone S-630 was found to be the most promising approach in terms of optimum CE and K _C. GFZ–β-CD (1:1) binary and ternary systems were prepared by kneading and lyophilization methods. The ternary systems clearly signified superiority over binary systems in terms of CE, solubility, K _C, and reduction in the formulation bulk. Optimized ternary system of GFZ–β-CD–Plasdone S-630 prepared by using lyophilization method indicated a significant improvement in intrinsic dissolution rate when compared with ternary kneaded system. Differential scanning calorimetry, X-ray diffraction, Fourier transform infrared, scanning electron microscopy, and proton nuclear magnetic resonance were carried out to characterize the binary and optimized ternary complex. The results suggested the formation of new solid phases, eliciting strong evidences of ternary inclusion complex formation between GFZ, β-CD, and Plasdone S-630, particularly for lyophilized products.     

Capillary operators—II

This work continues with an examination of capillary exchange models as operators, namely the operatorsO _k andK _αk relating extravascular and intravascular concentration to input for the Krogh cylinder model of a single capillary, a model basic to many organ models. Fundamental algebraic and analytic properties are presented: the operators belong to a commutative Banach algebra; an addition theorem holdsK _αk +K _βk =K _α+β,k ; the operatorK _αk has an inverse;K _αk ^-1 , (as an operator on LebesgueL _p space or on the locally integrable functions); partial derivatives are given forK _αk [f](t) andO _k [f](t) (sensitivity functions); and inequalities are established for the derivatives. Dominance relations between model curves are inferred. Error bound formulas are presented forK andO as bounds on ‖K _αk f-K _βl f‖ _p and ‖O _k f-O _l f‖ _p for allL _p . Consequent limitations on relative errors are shown. The implications for operators on a finite time interval are deduced. This work supported in part by PHS Grant Nos HL-19153 (SCOR and Pulmonary Vascular Disease) and HL-19370 at Vanderbilt University Medical School.     

Kinetic characterization of enhanced lipase activity on oil bodies

Enzyme access, kinetic behavior, and protein–protein interactions are critical for explaining reaction of the metabolites contained within the myriad compartments of biological systems. To explore these relationships, the reaction kinetics of oil bodies versus oil emulsions as substrates for lipolytic reactions were measured. The initial rate of hydrolysis for the oil body system was comparatively very low due to a brief latency period. However, the complete activation of the lipase at the interface resulted in an enzyme–membrane complex that was catalytically enhanced 3–15-fold over the emulsion system for substrate concentrations in the measured range of approximately 1–5.5 mM. This disparity is explained by the availability of substrate to the enzyme active site (defined as the availability parameter “A”) which varies between the two substrates by 40-fold. A simple hyperbolic kinetic mechanism is proposed with K _m replaced by the parameter, A, to account for this phenomenon, leading to a maximum rate of approximately 1450 IU/mg protein. The interaction is verified through separation of the enzyme–membrane complex which shows nearly double the activity towards an emulsified soybean oil substrate (activity ratio of 5:3) when compared to the native enzyme.     

Stomatal conductance and leaf water potential responses to hydraulic conductance variation in <Emphasis Type="Italic">Pinus pinaster</Emphasis> seedlings

In this study, tree hydraulic conductance (K _tree) was experimentally manipulated to study effects on short-term regulation of stomatal conductance (g _s), net photosynthesis (A) and bulk leaf water potential (Ψ_leaf) in well watered 5–6 years old and 1.2 m tall maritime pine seedlings (Pinus pinaster Ait.). K _tree was decreased by notching the stem and increased by progressively excising the root system and stem. Gas exchange was measured in a chamber at constant irradiance, vapour pressure deficit, leaf temperature and ambient CO₂ concentration. As expected, we found a strong and positive relationship between g _s and K _tree (r = 0.92, P = 0.0001) and between A and K _tree (r = 0.9, P = 0.0001). In contrast, however, we found that the response of Ψ_leaf to K _tree depended on the direction of change in K _tree: increases in K _tree caused Ψ_leaf to decrease from around −1.0 to −0.6 MPa, but reductions in K _tree were accompanied by homeostasis in Ψ_leaf (at −1 MPa). Both of these observations could be explained by an adaptative feedback loop between g _s and Ψ_leaf, with Ψ_leaf prevented from declining below the cavitation threshold by stomatal closure. Our results are consistent with the hypothesis that the observed stomatal responses were mediated by leaf water status, but they also suggest that the stomatal sensitivity to water status increased dramatically as Ψ_leaf approached −1 MPa.     

Identification of a specific inhibitor of nOGA — a caspase-3 cleaved O-GlcNAcase variant during apoptosis

O-Linked N-acetylglucosamine (O-GlcNAc) modification of serines/threonines on cytoplasmic proteins is a significant signal regulating cellular processes such as cell cycle, cell development, and cell apoptosis. O-GlcNAcase (OGA) is responsible for the removal of O-GlcNAc, and it thus plays a critical role in O-GlcNAc metabolism. Interestingly, OGA can be cleaved by caspase-3 into two fragments during apoptosis, producing an N-terminal fragment (1–413 a.a.), termed nOGA. Here, using 4-MU-GlcNAc (4-methylumbelliferyl 2-acetamido-2-deoxy-β-D-glucopyranoside) as substrate, we found that the nOGA fragment retains high glycosidase activity. To probe the role of nOGA in apoptosis, it is essential to develop a potent and specific nOGA inhibitor. However, many reported inhibitors active at nanomolar concentrations (including PUGNAc, STZ, GlcNAc-statin, and NAG-thiazoline) against full-length OGA were not potent for nOGA. Next, we screened a small triazole-linked carbohydrate library and first identified compound 4 (4-pyridyl-1-(2′-deoxy-2′-acetamido-β-D-glucopyranosyl)-1,2,3-triazole) as a potent and competitive inhibitor for nOGA. This compound shows 15-fold selectivity for nOGA (K _i = 48 μM) over the full-length OGA (K _i = 725 μM) and 10-fold selectivity over human lysosomal β-hexosaminidase A&B (Hex A&B) (K _i = 502 μM). These results reveal that compound 4 can be used as a potent and selective inhibitor for probing the role of nOGA in biological systems.     

Membrane responses to changes in the extracellular potassium concentration in isolated hippocampal pyramidal neurons

The responses of freshly isolated hippocampal pyramidal neurons to rapid, elevations of the external potassium concentration ([K⁺] _out ) were investigated using the whole-cell variation of a patch-clamp technique. An elevation of [K⁺] _out induced a two-phase inward current at the membrane potentials more negative than the reversal potential for K ions. This current consisted of a leakage, current and a time-dependent current (τ=40–50 msec at 21°C), the latter designated below asI _ΔK. It displayed first-order activation kinetics that showed neither voltage, nor concentration dependence. The amplitude of this current was determined by the external K⁺ concentration and increased with hyperpolarization. Voltage dependence ofI _ΔK measured within the range from −20 to −120 mV was similar to that for inward rectifier. Activation ofI _ΔK was utterly dependent on Na⁺; substitution of extracellular Na⁺ with choline chloride almost completely depressedI _ΔK.I _ΔK was absent in the cells freshly dissociated from the nodosal and dorsal root ganglia. This suggests that this earlier unrecognized current is instrumental in preserving densely packed hippocampal pyramidal neurons from sudden increases in [K⁺] _out and following spontaneous over-excitation. It prevents the neurons from responding to K⁺-induced depolarizations by slowing down potassium influx.     

Antioxidant activity of hydroxy derivatives of coumarin

The inhibition efficiency (antioxidant activity) of hydroxy derivatives of coumarin, such as esculetin, dicumarol, and fraxetin, was studied in the methemalbumin-H₂O₂-tetramethylbenzidine (TMB) pseudoperoxidase system at 20°C in a buffered physiological solution (pH 7.4) containing 6% DMF and 0.25% DMSO. The inhibitor’s efficiency was quantitatively characterized by the inhibition constants (K _i, μM) and the inhibition degree (%). The K _i values for esculetin, dicumarol, and fraxetin were 9.5, 15, and 26 μM, respectively. Esculetin and fraxetin inhibited pseudoperoxidase oxidation of TMB in a noncompetitive manner; dicumarol, in a mixed manner. The inhibiting activity of esculetin in peroxidase-catalyzed TMB oxidation at pH 6.4 is characterized by a K _i value equal to 1.15 μM, and the inhibition process is competitive. Esculetin was found to be the most effective antioxidant of plant origin among all derivatives previously studied in model biochemical systems.     

Auxin augments conductance of K+ inward rectifier in maize coleoptile protoplasts

Potassium is taken up by maize (Zea mays L.) coleoptile cells via a typical plant inward rectifier (K _ir ). Sufficient conductance of this channel is essential in order to maintain auxin-stimulated cell elongation. It was therefore investigated whether the activity of this channel is subject to direct or indirect control by this growth hormone. Patch-clamp measurements of whole coleoptile protoplasts revealed no appreciable effect of externally applied 10 μM or 100 μM α-naphthaleneacetic acid (NAA) on the activity of K _ir over test periods of ≥ 18 or ≥ 8 min, respectively. When, however, K _ir was recorded in the cell-attached configiuration and 10 μM NAA administered to the bath medium, the conductance of K _ir increased significantly in 13 out of 18 protoplasts over the control. This rise occurred at a fixed protoplast voltage after a lag period of less than 10 min and exhibited no voltage dependency. The absence of response to NAA of protoplasts in the whole-cell configuration indicates that auxin perception and channel control is linked via a soluble cytoplasmic factor and that this mediator is washed out or modified upon perfusion of the cytoplasm with pipette solution. To search for this expected diffusible factor the K _ir current was recorded before and after elevation of Ca²⁺ and H⁺ in the cytoplasm. In the whole-cell configuration the increase in Ca²⁺ from a nanomolar value to >1 μM by means of Ca²⁺-release from the caged precursor Na₂-DM-nitrophen left K _ir unaffected. The whole-cell K _ir conductance was also not affected upon addition of 10 mM Na⁺-acetate to the bath medium, an operation used to lower the cytoplasmic pH. This excludes a primary role for the known auxin-evoked rise in cytoplasmic Ca²⁺ and H⁺ in K _ir activity. We postulate that another, as yet unknown, mechanism mediates the auxin-evoked stimulation of the number of active K _ir channels in the plasma membrane. Received: 13 May 1998 / Accepted: 9 November 1998     

Pharmacological properties of the potassium-activated inward current in pyramidal hippocampal neurons

Elevation of the external potassium concentration induced a two-phase inward current in freshly isolated pyramidal hippocampal neurons. This current was voltage-dependent and demonstrated strong inward rectification. The current consisted of a leakage current and a time-dependent current (τ=40–50 msec at 21°C); the latter was designated asI _ΔK. As was shown earlier, K⁺ is a major charge carrier in the development of slow potassium-activated current. The pharmacological properties ofI _ΔK were studied using a patch-clamp technique.I _ΔK was completely blocked by external 10 mM TEA or 5 mM Ba²⁺ (IC₅₀=480±90mM) and exhibited low sensitivity to extracellular Cs⁺ (2 mM). This current was not affected by 1 mM 4-aminopyridine and was insensitive to a muscarinic agonist, carbachol (50 μM), and to 1 mM extracellular Cd²⁺. Elevation of external Ca²⁺ from 2.5 mM to 10 mM did not changeI _ΔK. Our data indicate that the pharmacological properties ofI _ΔK differ from those of other voltage-gated potassium currents, but more specific blockers must be used to make this evidence conclusive.     

Kinetic analysis of boron transport in Chara

The permeability of biological membranes to boric acid was investigated using the giant internodal cells of the charophyte alga Chara corallina (Klein ex Will. Esk. R.D. Wood). The advantage of this system is that it is possible to distinguish between membrane transport of boron (B) and complexing of B by plant cell walls. Influx of B was found to be rapid, with equilibrium between the intracellular and extracellular phases being established after approximately 24 h when the external concentration was 50 μM. The intracellular concentration at equilibrium was 55 μM, which is consistent with passive distribution of B across the membrane along with a small amount of internal complexation. Efflux of B occurred with a similar half-time to influx, approximately 3 h, which indicates that the intracellular B was not tightly complexed. The concentration dependence of short-term influx measured with ¹⁰B-enriched boric acid was biphasic. This was tentatively attributed to the operation of two separate transport systems, a facilitated system that saturates at 5 μM, and a linear component due to simple diffusion of B through the membrane. V _max and K _m for the facilitated transport system were 135 pmol m⁻²s⁻¹ and 2 μM, respectively. The permeability coefficient for boric acid in the Chara plasmalemma estimated from the slope of the linear influx component was 4.4 × 10⁻⁷cm s⁻¹ which is an order of magnitude lower than computed from the ether:water partition coefficient for B. Received: 14 August 2000 / Accepted: 16 September 2000     

Sodium-dependent and -independent Choline Uptake by Type II Epithelial Cells from Rat Lung

The uptake of ³H-labeled choline by a suspension of isolated type II epithelial cells from rat lung has been studied in a Ringer medium. Uptake was linear for 4 min at both 0.1 μm and 5.0 μm medium choline; at 5 μm, only 10% of the label was recovered in a lipid fraction. Further experiments were conducted at the low concentration (0.1 μm), permitting characterization of the properties of high-affinity systems. Three fractions of choline uptake were detected: (i) a sodium-dependent system that was totally inhibited by hemicholinium-3 (HC-3); (ii) a sodium-independent uptake, when Na⁺ was replaced by Li⁺, K⁺ or Mg²⁺, inhibited by HC-3; (iii) a residual portion persisting in the absence of Na⁺ and unaffected by HC-3. Choline uptake was sigmoidally related to the medium Na⁺ concentration. Kinetic properties of the uptake of 0.1 μm ³H-choline in the presence and absence of medium Na⁺ were examined in two ways. (a) Inhibition by increasing concentrations of unlabeled choline (0.5–100 μm) was consistent with the presence of two Michaelis-Menten-type systems in the presence of Na⁺; a Na⁺-dependent portion (a mean of 0.52 of the total) had a K _m for choline of 1.5 μm while K _m in the absence of Na⁺ (Li⁺ substituting) was 18.6 μm. (b) Inhibition by HC-3 (0.3–300 μm) gave K_i values of 1.7 μm and 5.0 μm HC-3 for the Na⁺-dependent and -independent fractions. The apparent K _m of the Na⁺-dependent uptake is lower than that reported previously for lung-derived cells and is in the range of the K _m values reported for high-affinity, Na⁺-dependent choline uptake by neuronal cells. Received: 18 February 1997/Revised: 7 December 1997     

Size-dependent variability of leaf and shoot hydraulic conductance in silver birch

Variation in leaf and shoot hydraulic conductance was examined on detached shoots of silver birch (Betula pendula Roth), cut from the lower third (shade leaves) and upper third of the crown (sun leaves) of large trees growing in a natural temperate forest stand. Hydraulic conductances of whole shoots (K _S), leaf blades (K _lb), petioles (K _P) and branches (i.e. leafless stem; K _B) were determined by water perfusion using a high-pressure flow meter in quasi-steady state mode. The shoots were exposed to irradiance of photosynthetic photon flux density of 200–250 μmol m⁻² s⁻¹, using different light sources. K _lb depended significantly (P < 0.001) on light quality, canopy position and leaf blade area (A _L). K _lb increased from crown base to tree top, in parallel with vertical patterns of A _L. However, the analysis of data on shade and sun leaves separately revealed an opposite trend: the bigger the A _L the higher K _lb. Leaf anatomical study of birch saplings revealed that this trend is attributable to enhanced vascular development with increasing leaf area. Hydraulic traits (K _S, K _B, K _lb) of sun shoots were well co-ordinated and more strongly correlated with characteristics of shoot size than those of shade shoots, reflecting their greater evaporative load and need for stricter adjustment of hydraulic capacity with shoot size. K _S increased with increasing xylem cross-sectional area to leaf area ratio (Huber value; P < 0.01), suggesting a preferential investment in water-conducting tissue (sapwood) relative to transpiring tissue (leaves), and most likely contributing to the functional stability of the hydraulic system, essential for fast-growing pioneer species.     

L-lysine Transport in Chicken Jejunal Brush Border Membrane Vesicles

The properties of l-lysine transport in chicken jejunum have been studied in brush border membrane vesicles isolated from 6-wk-old birds. l-lysine uptake was found to occur within an osmotically active space with significant binding to the membrane. The vesicles can accumulate l-lysine against a concentration gradient, by a membrane potential-sensitive mechanism. The kinetics of l-lysine transport were described by two saturable processes: first, a high affinity-transport system (K _mA= 2.4 ± 0.7 μmol/L) which recognizes cationic and also neutral amino acids with similar affinity in the presence or absence of Na⁺ (l-methionine inhibition constant K_iA, NaSCN = 21.0 ± 8.7 μmol/L and KSCN = 55.0 ± 8.4 μmol/L); second, a low-affinity transport mechanism (K_mB= 164.0 ± 13.0 μmol/L) which also recognizes neutral amino acids. This latter system shows a higher affinity in the presence of Na⁺ (K_iB for l-methionine, NaSCN = 1.7 ± 0.3 and KSCN = 3.4 ± 0.9 mmol/L). l-lysine influx was significantly reduced with N-ethylmaleimide (0.5 mmol/L) treatment. Accelerative exchange of extravesicular labeled l-lysine was demonstrated in vesicles preloaded with 1 mmol/L l-lysine, l-arginine or l-methionine. Results support the view that l-lysine is transported in the chicken jejunum by two transport systems, A and B, with properties similar to those described for systems b ^0,+ and y⁺, respectively. Received: 14 August 1995/Revised: 2 April 1996     

Expression,Purification, and Characterization of a Human Recombinant 17β-Hydroxysteroid Dehydrogenase Type 1 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the reaction of estrone with NADPH to form estradiol and NADP⁺, thereby regulating the biological activity of sex steroid hormones in a variety of tissues. Here, we present an efficient method for expressing and purifying human 17β-HSD1 from Escherichia coli. The expression vector pET28a/17β-HSD1 was constructed and transformed into Escherichia coli BL21(DE3) cells. We found that the active enzyme can be obtained by inducing 17β-HSD1 expression at 0.25 mM IPTG, 13°C for overnight. The protein is purified by single step Ni–NTA affinity chromatography and yields 2.8 mg/L of culture. The kinetic study shows V/E _t of (1.21 ± 0.05) × 10⁻²/s and K _estradiol of 0.8 μM in the oxidation of estradiol with NADP⁺ as cofactor at pH 9.3. The new bacterial expression system for recombinant 17β-HSD1 is useful for the easy purification of large amounts and will facilitate the functional study of this enzyme.     

Ion channels in guard cells of Arabidopsis thaliana (L.) Heynh.

Despite the availability of many mutants for signal transduction, Arabidopsis thaliana guard cells have so far not been used in electrophysiological research. Problems with the isolation of epidermal strips and the small size of A. thaliana guard cells were often prohibiting. In the present study these difficulties were overcome and guard cells were impaled with double-barreled microelectrodes. Membrane-potential recordings were often stable for over half an hour and voltage-clamp measurements could be conducted. The guard cells were found to exhibit two states. The majority of the guard cells had depolarized membrane potentials, which were largely dependent on external K⁺ concentrations. Other cells displayed spontaneous transitions to a more hyperpolarized state, at which the free-running membrane potential (E_m) was not sensitive to the external K⁺ concentration. Two outward-rectifying conductances were identified in cells in the depolarized state. A slow outward-rectifying channel (s-ORC) had properties resembling the K⁺-selective ORC of Vicia faba guard cells (Blatt, 1988, J Membr Biol 102: 235–246). The activation and inactivation times and the activation potential, all depended on the reversal potential (E_rev) of the s-ORC conductance. The s-ORC was blocked by Ba²⁺ (K_1/2 = 0.3–1.3mM) and verapamil (K_1/2 = 15–20 μM). A second rapid outward-rectifying conductance (r-ORC) activated instantaneously upon stepping the voltage to positive values and was stimulated by Ba²⁺. Inward-rectifying channels (IRC) were only observed in cells in the hyperpolarized state. The activation time and activation potential of this channel were not sensitive to the external K⁺ concentration. The slow activation of the IRC (t_1/2 ≈ 0.5 s) and its negative activation potential (V_threshold = −155 mV) resemble the values found for the KAT1 channel expressed in Saccharomyces cerevisiae (Bertl et al., 1995, Proc Natl Acad Sci USA 92: 2701–2705). The results indicate that A. thaliana guard cells provide an excellent system for the study of signal transduction processes. Received: 28 March 1996 / Accepted: 11 November 1996