The effect of aldosterone on the energetics of sodium transport in the frog skin

Uncertainty persists as to whether the stimulation of active sodium transport by aldosterone is attributable to effects on permeability or energetic factors. This question has been examined with the aid of a thermodynamic formulation in which the rate of both active sodium transport J_Na and O₂ consumption J_r are assumed to be linear functions of the electrical potential difference Δψ and the affinity A (negative free energy) of metabolic reaction. Previous studies have indicated constancy of a characteristic affinity on perturbation of Δψ, suggesting the possibility of its evaluation. In studies of paired frog skins the admnistration of aldosterone led to a significant increase in the short-circuit current I₀, a suggestive increase in the associated rate of O₂ consumption J_ro, and a significant increase in the ratio

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. If linearity obtains this ratio is equal to A. Depression of active sodium transport and the associated metabolism with amiloride, which depresses permeability, also results in an increase in the apparent affinity

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. The results indicate that aldosterone does not act simply by increasing the permeability or the number of transport units operating in parallel, but suggests that energetic factors are implicated as well.     

Thermodynamic analysis of active sodium transport and oxidative metabolism in toad urinary bladder.

Measurements of electrical current and oxygen consumption were carried out concurrently under voltage clamp conditions in 11 toad hemibladders. Inhibition of active transport with amiloride then permitted evaluation of the passive conductance and the rate of basal oxygen consumption Jbr, allowing the simultaneous determination of the rates of active sodium transport JaNa and suprabasal oxygen consumption Jsbr-JaNa and Jabr were linear functions of the electrical potential difference over a range of +/- 80 mV. This allowed the comprehensive application of a linear nonequilibrium thermodynamic formalism, leading to the evaluation of the affinity A (negative free energy) of the metabolic reaction driving transport, all phenomenological coefficients, and the degree of coupling q relating transport to metabolism. Values of A determined by two techniques were A1=56.0 +/- 5.8 and A2=58.2 +/- 6.5 kcal per mole. Values of q determined by two techniques agreed well and were less than 1, indicating incompleteness of coupling, and hence lack of fixed stoichiometry between Na transort and O2 consumption. The affinity and the electromotive force of sodium transport ENa are not closely correlated, reflecting the fact that ENa comprises both kinetic and energetic factors.     

Stimulation by aldosterone of a conductive chloride pathway in toad skin

As a rule, chloride movement (JC1-) across amphibian skin is considered to be passive; this is implied in fact for preparations incubated in Ringer's fluid, since short-circuit current (Isc) is the quantitative expression of net, active sodium transport (JNa+). The nature of the Cl- pathway(s) was investigated by incubating amphibian skin (mostly Bufo marinus) with Cl- present on the epithelial side only, and after blocking JNa+ by combined treatment with ouabain and amiloride. In such conditions, JCl- was found to be equal to (reversed) Isc; furthermore, when JCl- was "translated" in terms of conductance, gCl-, the latter accounted almost quantitatively for transepithelial conductance, g1. When residual intratissue (i.e. intracellular) electronegativity was eliminated by replacing Na+ with K+, JCl- was larger but Isc and JCl- were still found to reflect each other, and gCl- again accounted for most, if not all, of g1. JCl- in the opposite direction, as a result of Cl- being present only on the dermal side, was negligible, and g1 was very low. Thus, in the absence of sodium transport, when experimental conditions are such that a net inward JCl- obtains, the anion apparently flows only through (a) conductive pathway(s). Aldosterone is probably involved in the regulation of this pathway, as JCl- was much lower when toads were maintained in dilute saline than in water or on moist peat; so was the fraction of the apical surface corresponding to mitochondria-rich cells.     

Metabolic cost of sodium transport in toad urinary bladder.

The metabolic cost of active sodium transport was determined in toad bladder at different gradients of transepithelial potential. Deltapsi, by continuous and simultaneous measurements of CO2 production and of transepithelial electric current. Amiloride was used to block active sodium transport in order to assess the nontransport-linked, basal, production of CO2 and the passive permeability of the tissue. From these determinations active sodium transport, Jna, and suprabasal CO2 production, Jsb CO2, were calculated. Since large transients in Jna and Jsb CO2 frequently accompanied any abrupt change in deltapsi, steady state conditions were carefully defined. Some 20 to 40 min were required after a change in deltapsi before steady state of transport activity and of CO2 production were achieved. The metabolic cost of sodium transport proved to be the same whether the bladder expended energy moving sodium against a transepithelial electrical potential grandient of +50 mV or whether sodium was being pulled through "the active transport pathway" by an electrical gradient of -50 mV. In both cases the value of the ratio Jna/Jsb CO2 averaged some 20 sodium ions transported per molecule of CO2 produced. When the Na pump was blocked by 10(-2) M ouabain, the perturbations of the transepithelial electrical potential did not elicit changes of Jna nor, consequently of Jsb CO2. The independence of the ratio Jna/Jsb CO2 from deltapsi over the range+/-50 mV indicates a high degree of coupling between active sodium transport and metabolism.     

New aspects in the study of the mechanism of intestinal absorption of thiamine in rats

Intestinal absorption of thiamine was studied in rats using such metabolic inhibitors as ouabain, sodium azide and theophylline. These substances changed certain biochemical parameters of the mucosa in the small intestine affecting the level of thiamine transport across the intestinal epithelium. It was found that the intestinal transport of thiamine at lower concentration was an active process depending on the activity of Na, K, Mg-ATPase in the intestinal microvilli, and on the activity of mitochondrial processes. The used metabolic inhibitors increased the intestinal diffusion of thiamine at its higher concentration suggesting that these inhibitors changed membrane permeability by affecting enterocyte homeostasis. At both studied concentrations thiamine was rapidly absorbed from the digestive tract reaching the state of saturation which suggested a carrier character of this transport.     

Differences in c-AMP levels in epithelial cells from pelvic and pectoral regions of the toad skin

Arginine vasopressin (AVP) stimulated active Na+ transport (JNa+) and osmotic water flow (JH2O) across the pelvic skin but only JNa+ across the pectoral skin of the toad, Bufo woodhouseii. Isolated epithelial cells from the pelvic skin had a maximal c-AMP level of 11.16 pmoles/mg protein after 5 min of AVP treatment while that of pectoral skin was 3.64 pmoles/mg protein. The c-AMP level of both skin areas fell to unstimulated values after 20 min of AVP treatment; however, JH2O (pelvic skin) and JNa+ (pelvic and pectoral skin) remained elevated during 3 hr of treatment. Dibutyryl c-AMP and theophylline stimulated JH2O across the pelvic but not the pectoral skin. Maintaining toads in water for 12-24 hr resulted in a substantial lowering of JH2O across the pectoral skin which was not reversible by treatment with c-AMP and theophylline.     

The effect of T-2 toxin on active sodium transport across frog skin in the presence of ADH and amphotericin B

1. The effect of T-2 toxin on active sodium transport across frog skin both in the presence and in the absence of stimulants of sodium transport, such as Amphotericin B and ADH, was studied using the short circuit current technique with the following results. 2. T-2 toxin produces inhibition of active sodium transport in a dose-response correlation. 3. This effect is irreversible since the washing out of the tissue does not restore its functionality. This indicates that the micotoxin may cross the cellular membrane and act on the internal site. 4. ADH partially removes the inhibitory effect of T-2 toxin. 5. The increase of the sodium pool in the cell as determined by Amphotericin B does not reverse the inhibitory effect of T-2 toxin. 6. The biological significance of these data is discussed in regard to the possible effect of T-2 toxin on Na+, K+-ATPase activity either directly or by a reduction in the metabolic supply of substrates, or by a modified stoichiometry of the pump reaction.     

Calcium reduces the sodium permeability of luminal membrane vesicles from toad bladder. Studies using a fast-reaction apparatus

Regulation of the sodium permeability of the luminal membrane is the major mechanism by which the net rate of sodium transport across tight epithelia is varied. Previous evidence has suggested that the permeability of the luminal membrane might be regulated by changes in intracellular sodium or calcium activities. To test this directly, we isolated a fraction of the plasma membrane from the toad urinary bladder, which contains a fast, amiloride-sensitive sodium flux with characteristics similar to those of the native luminal membrane. Using a flow-quench apparatus to measure the initial rate of sodium efflux from these vesicles in the millisecond time range, we have demonstrated that the isotope exchange permeability of these vesicles is very sensitive to calcium. Calcium reduces the sodium permeability, and the half-maximal inhibitory concentration is 0.5 microM, well within the range of calcium activity found in cells. Also, the permeability of the luminal membrane vesicles is little affected by the ambient sodium concentration. These results, when taken together with studies on whole tissue, suggest that cell calcium may be an important regulator of transepithelial sodium transport by its effect on luminal sodium permeability. The effect of cell sodium on permeability may be mediated by calcium rather than by sodium itself.     

Anisotropic hydraulic permeability under finite deformation

The structural organization of biological tissues and cells often produces anisotropic transport properties. These tissues may also undergo large deformations under normal function, potentially inducing further anisotropy. A general framework for formulating constitutive relations for anisotropic transport properties under finite deformation is lacking in the literature. This study presents an approach based on representation theorems for symmetric tensor-valued functions and provides conditions to enforce positive semidefiniteness of the permeability or diffusivity tensor. Formulations are presented, which describe materials that are orthotropic, transversely isotropic, or isotropic in the reference state, and where large strains induce greater anisotropy. Strain-induced anisotropy of the permeability of a solid-fluid mixture is illustrated for finite torsion of a cylinder subjected to axial permeation. It is shown that, in general, torsion can produce a helical flow pattern, rather than the rectilinear pattern observed when adopting a more specialized, unconditionally isotropic spatial permeability tensor commonly used in biomechanics. The general formulation presented in this study can produce both affine and nonaffine reorientations of the preferred directions of material symmetry with strain, depending on the choice of material functions. This study addresses a need in the biomechanics literature by providing guidelines and formulations for anisotropic strain-dependent transport properties in porous-deformable media undergoing large deformations.     

Nonequilibrium Thermodynamic Analysis of the Coupling between Active Sodium Transport and Oxygen Consumption

Sodium transport and oxygen consumption have been simultaneously studied in the short-circuited toad skin. A constant stoichiometric ratio was observed in each skin under control condition (NaCl-Ringer's solution bathing both sides of the skin) and after block of sodium transport by ouabain. During alterations of sodium transport by removal and addition of K to the internal solution the stoichiometric ratio is constant although having a value higher than that observed in other untreated skins. The coupling between active sodium transport and oxygen consumption was studied after a theoretical nonequilibrium thermodynamic model. Studies were made of the influence of Na chemical potential difference across the skin on the rates of Na transport and oxygen consumption. A linear relationship was observed between the rates of Na transport and oxygen consumption and the Na chemical potential difference. Assuming the Onsager relationship to be valid, the three phenomenological coefficients which describe the system were evaluated. Transient increases in the rate of sodium transport and oxygen consumption were observed after a transitory block of sodium transport by removal of Na from the external solution. Cyanide blocks completely the rate of oxygen consumption in less than 2 min and the short-circuit current measured after that time decays exponentially with time, suggesting a depletion of ATP from a single compartment.     

Pharmacologic agents for the in vivo detection of vascular sodium transport defects in hypertension

Anatagonists to angiotensin, catecholamines, aldosterone, and vasopressin have long been used to help determine agonist roles in hypertension. We here call attention to a possible extension of this approach to detect, evaluate, and treat vascular sodium transport defects in hypertension. Two basic types of transport defects have been identified in the blood vessels of hypertensive animals, increased sodium permeability and decreased sodium pump activity. Intravenous injection of 6-iodo-amiloride, a sodium channel blocker and vasodilator, produces an immediate and sustained decrease in blood pressure in two genetic models of hypertension characterized by increased permeability of the vascular smooth muscle cell membrane to sodium (Okamoto spontaneously hypertensive rat, Dahl salt sensitive rat), whereas it produces only a transient fall in arterial pressure in two renal models of hypertension having normal sodium permeability in vascular smooth muscle cells (reduced renal mass-saline rat, one-kidney, one clip rat). Canrenone, a metabolic product of spironolactone which can compete with oubain for binding to Na+,K+-ATPase at the digitalis receptor site, decreases blood pressure in a low renin, volume expanded model of hypertension which has been shown to have depressed sodium pump activity in arteries and increased sodium pump inhibitor in plasma (reduced renal mass-saline rat) but has no effect on blood pressure in a genetic model of hypertension which has been shown to have increased sodium pump activity secondary to increased sodium permeability (spontaneously hypertensive rat). Thus, a sodium channel blocker and a competitor to ouabain binding can detect and determine the functional significance of sodium transport defects in the blood vessels of intact hypertensive animals. Studies in red and white blood cells suggest that similar defects may exist in the blood vessels of hypertensive humans. Thus, this approach, probing for vascular transport defects in the intact animal, may ultimately also be useful in the clinical setting.     

Lung edema clearance: 20 years of progress: invited review: alveolar edema fluid clearance in the injured lung.

Resolution of pulmonary edema involved active transepithelial sodium transport. Although several of the cellular and molecular mechanisms involved are relatively well understood, it is only recently that the regulation of these mechanisms in injured lung are being evaluated. Interestingly, in mild-to-moderate lung injury, alveolar edema fluid clearance is often preserved. This preserved or enhanced alveolar fluid clearance is mediated by catecholamine-dependent or -independent mechanisms. This stimulation of alveolar liquid clearance is related to activation or increased expression of sodium transport molecules such as the epithelial sodium channel or the Na(+)-K(+)-ATPase pump and may also involve the cystic fibrosis transmembrane conductance regulator. When severe lung injury occurs, the decrease in alveolar liquid clearance may be related to changes in alveolar permeability or to changes in activity or expression of sodium or chloride transport molecules. Multiple pharmacological tools such as beta-adrenergic agonists, vasoactive drugs, or gene therapy may prove effective in stimulating the resolution of alveolar edema in the injured lung.     

Control analysis of metabolic networks. 2. Total differentials and general formulation of the connectivity relations

The mathematical background of the connectivity relations of metabolic control theory is analysed. The connectivity relations are shown to reflect general properties of total differentials of reaction rate vi, flux J, and metabolite concentration Xj. Connectivity relations hold for any metabolic network in which all vi are homogeneous functions of enzyme concentration Ei. This notion allows established algebraic methods to be used for the formulation of connectivity relations for metabolic systems in which numerous constraints are imposed on metabolite concentrations. A general procedure to derive connectivity relations for such metabolic systems is given. To encourage a broader audience to apply control theory to physiological systems, an easy-to-use graphical procedure is derived for formulating connectivity relations for biochemical systems in which no metabolite is involved in more than one constraint.     

Coupling between the bacteriorhodopsin photocycle and the protonmotive force in Halobacterium halobium cell envelope vesicles. III. Time-resolved increase in the transmembrane electric potential and modeling of the associated ion fluxes.

Bacteriorhodopsin functions as an electrogenic, light-driven proton pump in Halobacterium halobium. In cell envelope vesicles, its photocycle kinetics can be correlated with membrane potential. The initial decay rate of the M photocycle intermediate(s) decreases with increasing membrane potential, allowing the construction of a calibration curve. The laser (592.5 nm) was flashed at various time delays following the start of background illumination (592 +/- 25 nm) and transient absorbance changes at 418 nm monitored in cell envelope vesicles. The vesicles were loaded with and suspended in either 3 M NaCl or 3 M KCl buffered with 50 mM HEPES at pH 7.5 and the membrane permeability to protons modified by pretreatment with N,N'-dicyclohexylcarbodiimide. In each case the membrane potential rose with a halftime of approximately 75 ms. The steady-state potential achieved depends on the cation present and the proton permeability of the membrane, i.e., higher potentials are developed in dicyclohexylcarbodiimide treated vesicles or in NaCl media as compared with KCl media. The results are modeled using an irreversible thermodynamics formulation, which assumes a constant driving reaction affinity (Ach) and a variable reaction rate (Jr) for the proton-pumping cycle of bacteriorhodopsin. Additionally, the model includes a voltage-gated, electrogenic Na+/H+ antiporter that is active when vesicles are suspended in NaCl. Estimates for the linear phenomenological coefficients describing the overall proton-pumping cycle (Lr = 3.5 X 10(-11)/mol2/J X g X s), passive cation permeabilities (LHu = 2 X 10(-10), LKu = 2.2 X 10(-10), LNau = 1 X 10(-11)), and the Na+/H+ exchange via the antiporter (Lex = 5 X 10(-11)) have been obtained.     

Isotope Flows and Flux Ratios in Biological Membranes

Precise evaluation of permeability of biological tissues is often prevented by imprecise knowledge of operative forces. This problem has been approached by analysis of fluxes of isotopic species applied to opposite surfaces of a membrane. A simple and rather general flux ratio equation has been derived which may permit evaluation of membrane permeability, even without knowledge of forces, or of the nature of active transport processes. Permeability as thus defined should be insensitive to coupled flows, either of other species or of metabolism. In appropriate circumstances application of the equation may permit evaluation of the contributions of the various processes to the transport of the examined species. Composite series membranes would be expected to obey the unmodified general equation. Heterogeneous parallel pathways would alter the relation in a predictable manner. The effect of isotope interaction is specifically incorporated. The formulation is applied to consideration of energetics of active transport.     

Metabolic consequences of limited substrate anion permeability in brown fat mitochondria from a hibernator,the golden hamster

Brown fat mitochondria obtained from a hibernator, the golden hamster, were investigated in order to elucidate the significance of membrane permeability for metabolic functioning at different temperatures. The mitochondria were shown to have active permeases for phosphate and pyruvate, but very poorly developed permeases for di- and tricarboxylate substrate anions. This was shown with both osmotic swelling techniques and respiration-driven uptake studies. It was shown that the very limited malate permeation observed was compatible with it being a non-carrier-mediated diffusion process. The role of malate transport in supporting fatty-acid oxidation in vitro as a function of temperature was studied in detail. The results support our earlier suggestion that physiologically pyruvate carboxylase probably functions to generate oxaloacetate when high concentrations of condensing partner are needed during thermogenesis. They may also explain earlier observations that acetate was produced from palmitoyl-carnitine at low temperatures even when malate was present; this is here shown to be due to the limited malate permeability at these low temperatures. Thus, even at the body temperature of the hibernating hamster (4–5°C), brown fat is probably able to combust fatty acids totally.     

Properties of solution of the diffusion-reaction equation

An arbitrary set of chemical reactions is considered to occur among chemical speciesX _i . In a closed uniform reaction system certain linear combinations of the concentrations of theX _i are constants. The general construction of all such linear combinations with non-negative coefficients is given in terms of the molecular formulae for theX _i . It is shown that to each such linear combination there corresponds another which is a harmonic function when the reactions take place in an open spatially distributed stationary reaction system of arbitrary shape. Under the usual boundary conditions these harmonic functions are constants. With some restrictions upon the diffusion and permeability coefficients these constants are evaluated. This evaluation is the basis for relations between the total concentration of a given chemical group (e.g., the sum of the concentrations of a free molecule, or ion, and its various bound forms) in the reaction system, and in the surrounding medium. The bearing of these relations on apparent active transport is noted and illustrated.     

Resolution of Pump and Leak Components of Sodium and Potassium Ion Transport in Human Erythrocytes

Further support for the pump-leak concept was obtained. Net transport was resolved into pump and leak components with the cardiac glycoside, ouabain. The specificity of ouabain as a pump inhibitor was demonstrated by its ineffectiveness when the pump was already inhibited by lack of one of the three pump substrates, sodium ion, potassium ion, or adenosine triphosphate. In the presence of ouabain the rates of passive transport of sodium and potassium ions changed almost in proportion to changes in their extracellular concentrations when one ion was exchanged for the other. In the presence of ouabain and at the extracellular concentrations which produced zero net transport, the ratio of potassium ions to sodium ions was 1.2-fold higher inside the cells than outside. This finding was attributed to a residual pump activity of less than 2% of capacity. The permeability to potassium ions was 10% greater than the permeability to sodium ions. A test was made of the independence of pump and leak. Conditions were chosen to change the rate through each pathway separately or in combination. When both pathways were active, net transport was the sum of the rates observed when each acted separately. A ratio of three sodium ions pumped outward per two potassium ions pumped inward was confirmed.     

Comparison of the effects of dDAVP and AVP on the sodium transport in the frog skin

The effect of 1-deamino-8-D-arginine-vasopressin, dDAVP, the synthetic analogue of vasopressin, upon the active sodium transport across the frog skin was studied using standard microelectrode technique and compared with the effect of synthetic arginine-vasopressin, AVP. dDAVP applied to the basolateral side of the epithelium stimulated the active sodium transport as reflected by the increase of short-circuit current, Isc, and transepithelial electrical potential difference, Voc. Potential difference across both the apical, Vo, and the basolateral, Vi, cell membranes decreased. The driving force of transepithelial sodium transport, ENa, did not change. The transepithelial electrical resistance, Rt, ohmic resistance of the active sodium transport, RNa, and apical cell membrane resistance, Ro, rapidly decreased, while the resistance of the basolateral cell membrane, Ri, and the resistance of the shunt pathway, Rs, remained unchanged. It is concluded that dDAVP primarily increases sodium permeability of the apical cell membrane which subsequently stimulates sodium pump activity. This action is similar to that of AVP.     

Metabolic regulation of apical sodium permeability in toad urinary bladder in the presence and absence of aldosterone

In the present study, further evidence was adduced for energy-dependent regulation of passive apical transport of Na in toad bladder epithelium. In potassium-depolarized preparations studied by current-voltage analysis, additions of pyruvate or glucose to the media of substrate-depleted bladders evoked proportionate increases in the transepithelial Na current and in apical Na permeability. These responses were large in aldosterone pretreated hemibladders and almost absent in the aldosterone-depleted preparations or when hormonal action was blocked by spironolactone or cycloheximide. The substrate-induced increases in apical Na permeability were fully reversed by appropriate metabolic inhibitors, i.e. 2-deoxyglucose and oxythiamine. Moreover, the inhibitory effect of 2-deoxyglucose was bypassed by the addition of pyruvate to the serosal medium. Thus apical Na permeability is clearly sensitive to the supply of cellular energy. The possibility that changes in intracellular free Na activity may mediate metabolic regulation of apical Na permeability was evaluated by prolonged exposure to Na-free mucosal and serosal media, with and without inhibition of the Na/K-pump by ouabain. The stimulatory and inhibitory effects of pyruvate, 2-deoxyglucose and oxythiamine on Na currents and Na conductances were preserved under these circumstances. Furthermore, reduction of serosal Ca to a minimal level of 3 microM, was without effect on the response to metabolic inhibition. These experiments demonstrate the existence of Na-independent metabolic regulation of apical Na transport and imply that neither basal-lateral nor mitochondrial Na/Ca exchange is required for this regulatory process under the imposed conditions. The possibility that a Na-independent, Ca transport mechanism in mitochondria or endoplasmic reticulum may be involved in metabolic regulation of apical Na transport, however, remains to be evaluated.