Energetics of Sodium Transport in Frog Skin : II. The effects of electrical potential on oxygen consumption

Studies were made of the dependence of the rate of oxygen consumption, J_r, on the electrical potential difference, Δψ, across the frog skin. After the abolition of sodium transport by ouabain the basal oxygen consumption was independent of Δψ. In fresh skins J_r was a linear function of Δψ over a range of at least ±70 mv. Treatment with aldosterone stimulated the short-circuit current, I_o, and the associated rate of oxygen consumption, J_ro, and increased their stability; linearity was then demonstrable over a range of ±160 mv. Brief perturbations of Δψ (±30–200 mv) did not alter subsequent values of I_o. Perturbations for 10 min or more produced a "memory" effect both with and without aldosterone: accelerating sodium transport by negative clamping lowered the subsequent value of I_o; positive clamping induced the opposite effect. Changes in J_ro were more readily detectable in the presence of aldosterone; these were in the same direction as the changes in I_o. The linearity of J_r in Δψ indicates the validity of analysis in terms of linear nonequilibrium thermodynamics—brief perturbations of Δψ appear to produce no significant effect on either the phenomenological coefficients or the free energy of the metabolic driving reaction. Hence it is possible to evaluate this free energy.     

A comparison of the effects of ouabain and 2-deoxy-d-glucose on the thermodynamic variables of the frog skin

Previous studies support the validity of a linear thermodynamic formalism relating the rates of active Na⁺ transport and oxygen consumption J_r to the electrical potential difference ΔΨ an the affinity α (negative free energy) of the metabolic driving reaction. The formulation was further tested in paired control and experimental hemiskins by the use of two inhibitors of Na⁺ transport. Ouabain, a specific inhibitor of the Na⁺ pump, might be expected to diminish the dependence of J_r on ΔΨ without affecting α, whereas 2-deoxy-d-glucose, a competitive inhibitor of glucose metabolism, should be expected to diminish α. Both inhibitors were used at concentrations adequate to depress Na⁺ transport (i.e. short-circuit current J_o) to some 50°_o of control level. Measurements were made of I_o and $\text{d}\text{J}{}_{\mathrm{<mtext>r</mtext>}}\text{d}\text{(ΔΨ)}$, and the apparent value of the affinity α_app was calculated according to the thermodynamic formulation. Ouabain depressed $\text{d}\text{J}{}_{\mathrm{<mtext>r</mtext>}}\text{d}\text{(ΔΨ)}$ without affecting α_app whereas 2-deoxy-d-glucose depressed α_app without affecting $\text{d}\text{J}{}_{\mathrm{<mtext>r</mtext>}}\text{d}\text{(αΨ)}$. The demonstration of these effects indicates the utility of the formalism.     

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.     

Energetics of Active Transport Processes

Discussions of active transport usually assume stoichiometry between the rate of transport J₊ and the metabolic rate J_r. However, the observation of a linear relationship between J₊ and J_r does not imply a stoichiometric relationship, i.e., complete coupling. Since coupling may possibly be incomplete, we examine systems of an arbitrary degree of coupling q, regarding stoichiometry as a limiting case. We consider a sodium pump, with J₊ and J_r linear functions of the electrochemical potential difference, -X₊, and the chemical affinity of the metabolic driving reaction, A. The affinity is well defined even for various complex reaction pathways. Incorporation of a series barrier and a parallel leak does not affect the linearity of the composite observable system. The affinity of some region of the metabolic chain may be maintained constant, either by large pools of reactants or by regulation. If so, this affinity can be evaluated by two independent methods. Sodium transport is conveniently characterized by the open-circuit potential (Δψ)_I=0 and the natural limits, level flow (J₊)_X+=0, and static head X⁰₊ = (X₊)_J+=0. With high degrees of coupling -X⁰₊/F approaches the electromotive force E_Na (Ussing); -X⁰₊/F cannot be identified with ((RT/F) ln f)_X+=0, where f is the flux ratio. The efficiency η = -J₊X₊/J_rA is of significance only when appreciable energy is being converted from one form to another. When either J₊ or -X₊ is small η is low; the significant parameters are then the efficacies ε_J+ = J₊/J_rA and ε_X+ = -X₊/J_rA, respectively maximal at level flow and static head. Leak increases both J₊ and ε_J+ for isotonic saline reabsorption, but diminishes -X⁰₊ and ε_X♀. Electrical resistance reflects both passive parameters and metabolism. Various fundamental relations are preserved despite coupling of passive ion and water flows.     

Sodium transport and oxygen consumption in toad bladder: A thermodynamic approach

The relationship between active sodium transport and oxygen consumption was investigated in toad urinary bladder exposed to identical sodium-Ringer's solution at each surface, while controlling the transepithelial electrical potential difference Δψ. Rates of sodium transport and oxygen consumption were measured simultaneously, both in the short-circuited state (Δψ = 0) and when Δψ was varied. Under short-circuit conditions, when the rates of active sodium transport changed spontaneously or were depressed with amiloride, the ratio of active sodium transport to the estimated suprabasal oxygen consumption Na⁺/O₂ was constant for each tissue, but varied among different tissues. Only when Δψ was varied did the ratio Na⁺/dO₂ change with the rate of active sodium transport; under these circumstances dNa⁺/dO₂ was constant but exceeded the ratio measured at short-circuit [(Na⁺/O₂)_Δψ=0]. This suggests that coupling between transport and metabolism is incomplete. The results are analyzed according to the principles of nonequilibrium thermodynamics, and interpreted in terms of a simple model of the transepithelial sodium transport system.     

On-line continuous measurement of the oxygen consumption rate in mammalian cell culture

A method for on-line, continuous measurement of the oxygen consumption rate (Io₂) by mammalian cells on a microcarrier was developed and its reliability investigated. Utilizing the periodic dissolved oxygen (DO) fluctuation in the normal on-off control of DO, on-line, continuous measurement of Io₂ was carried out in which real-time estimation of the DO saturation concentration was made by measuring the gas-phase pressure and the gas-phase oxygen concentration. It was found that the continuously measured Io₂ value was quantitatively exact and could be applied commercially using the oxidation reaction of glucose by glucose oxidase.     

Coordination chemistry of higher oxidation states. Part 21. Platinum-195 NMR studies of platinum(II) and platinum(IV) complexes of bi- and multi-dentate phosphorus,arsenic and sulphur ligands

195-Platinum NMR spectra are reported for a series of complexes of bidentate ligands [Pt(LL)X₄] (X=Cl, Br; LL=diphosphine, diarsine, dithioether, diselenoether), [Pt(Me₂PCH₂CH₂PMe₂)₂X₂]X₂, [Pt(o-C₆H₄(AsMe₂)₂)₂X₂]X₂, and for the Pt(II) analogues. The trends in chemical shifts δ(Pt) and ¹J(PtP), ¹J(PtSe) coupling constants are discussed, and used to establish the nature of the solution species obtained by oxidation of Pt(II) complexes of some multidentate phosphorus and arsenic ligands. The [Pt(LL)I₄] materials are shown to exist as [Pt^II(LL)I₂] in dimethylsulphoxide solution, but [Pt(o-C₆H₄(AsMe₂)₂)₂I₂]²⁺ is a genuine Pt(IV) iodo-complex.     

The Sodium-Potassium Exchange Pump: Relation of Metabolism to Electrical Properties of the Cell: I. Theory

The Na-K exchange pump is represented as a net stoichiometrically coupled reaction, r, involving ATP, Na⁺, and K⁺, and is located in the active region of the cell membrane. The reaction rate is J_r = L_rr (-ΔF_r), where ΔF_r is the free energy change of the reaction. ΔF_r includes membrane potential ø₂ in the absence of 1:1 coupling between Na⁺ and K⁺, and the reaction rate is potential dependent under these conditions. At the same time the pump will produce a potential H which is the difference between membrane potential and the diffusion potential as calculated with constant field assumptions. In the absence of 1:1 coupling, the pump is electrogenic. The feedback relation between reaction rate and membrane potential makes the membrane resistance in the presence of the pump less than or equal to the resistance in its absence, at the same membrane potential. H depends on stoichiometry, reaction rate, and passive ionic conductances. Experimental verification of the model will depend on the accuracy of permeability determinations. Dissipation and efficiency of transport can be calculated also.     

Respiration and sodium transport in rabbit urinary bladder

Respiration of rabbit urinary bladder was measured in free-floating pieces and in short-circuited pieces mounted in an Ussing chamber. Ouabain, amiloride, and potassium-free saline inhibited respiration approx. 20%; sodium-free saline depressed respiration approx. 40–50%. The coupling ratio between respiration and transport in short-circuited tissues was about two sodium ions per molecule O₂. Chloride-free saline depressed mean oxygen consumption 21% in free-floating tissue pieces; 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS) and furosemide had no effect. The effect of chloride-free saline in short-circuited tissues was variable; in tissues with low transport rates, respiration was stimulated about 21% while in tissue with high transport rates respiration was reduced about 24%. Nystatin and monensin, both of which markedly increase the conductance of cell membranes with a concomitant increase in sodium entry, stimulated respiration. These data indicate that 50–60% of the total oxygen consumption is not influenced by sodium, 20–25% is linked to (Na⁺ + K⁺)-ATPase transport, while the remaining 25–30% is sodium-dependent but not ouabain-inhibitable.     

The effects of external potassium and long duration voltage conditioning on the amplitude of sodium currents in the giant axon of the squid, Loligo pealei

Giant axons were voltage-clamped in solutions of constant sodium concentration (230 mM) and variable potassium concentrations (from 0 to 210 mM). The values of the peak initial transient current, I_p, were measured as a function of conditioning prepulse duration over the range from less than 1 msec to over 3 min. Prepulse amplitudes were varied from E _m = -20 mv to E _m = -160 mv. The attenuation of the I_p values in high [K_o] was found to vary as a function of time when long duration conditioning potentials were applied. In both high and low [K_o], I_p values which had reached a quasi-steady—state level within a few milliseconds following a few milliseconds of hyperpolarization were found to increase following longer hyperpolarization. A second plateau was reached with a time constant of about 100–500 msec and a third with a time constant in the range of 30 to 200 sec. The intermediate quasi-steady—state level was absent in K-free ASW solutions. Sodium inactivation curves, normalized to I _pmax values obtained at either the first or second plateaus, were significantly different in different [K_o]. The inactivation curves, however, tended to superpose after about 1 min of hyperpolarizing conditioning. The time courses and magnitudes of the intermediate and very slow sodium conductance restorations induced by long hyperpolarizing pulses are in agreement with those predicted from the calculated rates and magnitudes of [K⁺] depletion in the space between the axolemma and the Schwann layer.     

Rates of oxygen consumption by Thais (Nucella) Lapillus (L.)

Oxygen consumption by Thais varied seasonally with higher values in summer than in winter. This seasonal difference was due in part to the effects of temperature and in part to those of feeding. During feeding, rates of oxygen consumption were high, but declined in the period between meals. There was little evidence of acclimation of oxygen consumption to changes in temperature; low (winter) rates of consumption were more sensitive to increases in temperature than were high (summer) rates. A polynomial expression, including terms for temperature and ‘time since last meal’, was derived for the constant a′ in the allometric equation relating oxygen consumption (_o2) to dry body weight: _o2 = a′.W^0.511.     

Characterization of mitochondrial bioenergetic functions between two forms of Leishmania donovani – a comparative analysis

Leishmaniasis is a growing health problem in many parts of the world partly due to drug resistance of the parasite. This study reports on the fisibility of studying mitochondrial properties of two forms of wild-type L. donovani through the use of selective inhibitors. Amastigote forms of L. donovani exhibited a wide range of sensitivities to these inhibitors. Mitochondrial complex II inhibitor thenoyltrifluoroacetone and F_oF₁-ATP synthase inhibitors oligomycin and dicyclohexylcarbodiimide were refractory to growth inhibition of amastigote forms, whereas they strongly inhibited the growth of promastigote forms. This result indicated that complex II and F_oF₁-ATP synthase were not functional in amastigote forms suggesting the presence of attenuated oxidative phosphorylation in the mitochondria of amastigote forms. In contrast, mitochondrial complex I inhibitor rotenone and complex III inhibitor antimycin A inhibited cellular multiplication and substrate level phosphorylation in amastigote forms, suggesting the role of complex I and complex III for the survival of amastigote forms. Further we studied the mitochondrial activities of both forms by measuring oxygen consumption and ATP production. In amastigote form, substantial ATP formation by substrate level phosphorylation was observed in NADPH-fumarate, NADH-fumarate, NADPH-pyruvate and NADH-pyruvate redox couples. None of the redox couple generated ATP formation was inhibited by F_oF₁-ATP synthase inhibitor oligomycin. Therefore, we may conclude that there are significant differences between these two forms of L. donovani in respect of mitochondrial bioenergetics. Our results demonstrated bioenergetic disfunction of amastigote mitochondria. Therefore, these alterations of metabolic functions might be a potential chemotherapeutic target.     

TEMPERATURE AND CRITICAL ILLUMINATION FOR REACTION TO FLICKERING LIGHT : V. XIPHOPHORUS,PLATYPOECILIUS, AND THEIR HYBRIDS

For the teleosts Xiphophorus montezuma, Platypoecilius maculatus, and their F ₁ hybrids the temperature characteristics (µ in Arrhenius'' equation) are the same for the shift of the low intensity and the high intensity segments of the respective and different flicker response contours (critical intensity I as a function of flash frequency F, with light time fraction constant, at 50 per cent). The value of µ is 12,500 calories or a very little less, over the range 12.5 to 36°. This shows that 1/I can be understood as a measure of excitability, with F fixed, and that the excitability is governed by the velocity of a chemical process common to both the classes of elements represented in the duplex performance curve (rods and cones). It is accordingly illegitimate to assume that the different shapes of the rod and cone branches of the curves are determined by differences in the chemical mechanisms of excitability. It is also forbidden to assume that the differing form constants for the homologous segments in the curves for two forms (X. and P.) are the reflections of a difference in the chemical factors of primary excitability. These differences are determined by statistical factors of the distribution of excitabilities among the elements implicated in the sensory effect vs. intensity function, and are independent of temperature and of the temperature characteristic. It must be concluded that the physicochemical nature of the excitatory process cannot be deduced from the shape of the performance contour. The form constants (σ''_{log I} and F_max.) for F vs. log I are specifically heritable in F ₁, although µ is here the same as for X. and P. In an intergeneric cross one cannot in general expect Mendelian simplicity of segregation in subsequent generations, and in the present case we find that F ₂ individuals are indistinguishable from F ₁, both as regards F vs. log I and as regards the variation of I within a group of 17 individuals. The result in F ₂ definitely shows, however, that certain specific statistical form constants for the F-log I contour are transmissible in inheritance. It is pointed out that there thus is provided an instance in which statistical (distribution) factors in performance characteristics involving the summating properties of assemblages of cellular units are heritable in a simple manner without the implication of detectable differences in chemical organization of the units involved. This has an important bearing upon the logic of the theory of the gene.     

Non-Linear Current-Potential Relations in an Axon Membrane

The membrane current density, I_m, in the squid giant axon has been calculated from the measured external current applied to the axon, I_o, by the equation See PDF for Equation where V_m is the membrane potential under the current electrode and r₁ and r₂ are the external and internal longitudinal resistances. The original derivation of this equation included in one step an assumption of a linear relation between I_m and V_m. It is shown that the same equation can be obtained without this restricting assumption.     

Cation binding to beta-casein. A comparison of electrostatic models

The binding of cations of β-casein at pH 6.6 was considered previously. Available for three sodium concentiations, I = 0.04, 0.08, or 0.16 M are: [1] proton releases between I and [2] for each I, as calcium activity is increased, correlated sequences of monomer net charge, proton release, site bound calcium and protein Solvation- Models for ion binding are examined. Critical considerations are the intrinsic binding constants between hydrogen[H], calcium[Ca] and sodium[Na] ions and phosphate[P] and caiboxyIate[C] sites, and the effects of electrostatic interaction between sites as influenced by spatial fixed charge distribution, ionic strength and dielectric constant [D]. Anticipated intrinsic binding constants are k_H,P^o = 3 × 10⁶, k_Ca,P^o = 120, k_Na,P^o = 1, k_H,C^o = 7 × 10⁴ and k_Ca,C^o = 5.6Distributed charge models, either surface or volume, are inadequate since any reasonable monomer size yields fixed charge densities requiring k_H,P^o and k_Ca,C^o which are too low when the maximum in D is 75. Also, with increasing calcium binding, calculated proton release is only 0.4 to 0.5 of that observed.Discrete charge models accept anticipated k^o and yield calculated sequences of calcium binding and proton release which are in good agreement with those observed provided that: (1) using the known amino acid sequence of the phosphate-containing acidic peptide portion of the molecule, pep tide fixed charge is distributed at the lowest I so as to minimize electrostatic free energy; (2) in the region of fixed charge, D is approximately 5; (3) the distances between peptide fixed charges decrease with increasing ionic strength or calcium binding and (4) while protein is in solution, the acidic peptide and the remainder of the molecule are essentially electrostatically independent.     

CRITICAL ILLUMINATION AND FLICKER FREQUENCY,AS A FUNCTION OF FLASH DURATION: FOR THE SUNFISH

From the relations between critical illumination in a flash (I_m) and the flash frequency (F) for response of the sunfish to visual flicker when the proportion of light time to dark time (t_L/t_D) in a flicker cycle is varied at one temperature (21.5°) the following results are obtained: At values of t_L/t_D between 1/9 and 9/1 the F - log I_m curves are progressively shifted toward higher intensities and lower F_max.. F_max. is a declining rectilinear function of the percentage of the flash cycle time occupied by light. The rod and the cone portions of the flicker curve are not shifted to the same extent. The cone portion and the rod region of the curve are each well described by a probability integral. In terms of F as 100 F/F_max. the standard deviation of the underlying frequency distribution of elemental contributions, summed to produce the effect proportional to F, is independent of t_L/t_D. The magnitude of log I_m at the inflection point (r''), however, increases rectilinearly with the percentage light time in the cycle. The proportionality between I_m and σ_II1 is independent of t_L/t_D. These effects are interpreted as consequences of the fact that the number of elements of excitation available for discrimination of flicker is increased by increasing the dark interval in a flash cycle. Decreasing the dark interval has therefore the same kind of effect as reducing the visual area, and not that produced by decreasing the temperature.     

Sodium transport and oxygen consumption in toad bladder. A thermodynamic approach.

The relationship between active sodium transport and oxygen consumption was investigated in toad urinary bladder exposed to identical sodium-Ringer's solution at each surface, while controlling the transepithelial electrical potential difference delta phi. Rates of sodium transport and oxygen consumption were measured simultaneously, both in the short-circuited state (delta phi = 0) and when delta phi was varied. Under short-circuit conditions, when the rates of active sodium transport changed spontaneously or were depressed with amiloride, the ratio of active sodium transport to the estimated suprabasal oxygen consumption Na/O2 was constant for each tissue, but varied among different tissues. Only when delta phi was varied did the ratio Na+/O2 change with the rate of active sodium transport; under these circumstances dNa+/dO2 was constant but exceeded the ratio measured at short-circuit [(Na+/O2)delta phi = 0[. This suggests that coupling between transport and metabolism is incomplete. The results are analyzed according to the principles of nonequilibrium thermodynamics, and intepreted in terms of a simple model of the transepithelial sodium transport system.     

TEMPERATURE AND CRITICAL ILLUMINATION FOR REACTION TO FLICKERING LIGHT : IV. ANAX NYMPHS

At fixed flash frequency (_F = 20, _F = 55) and with constant light time fraction (50 per cent) in the flash cycle, the critical illumination I for response of Anax nymphs to visual flicker falls continuously as the temperature rises. The temperature characteristic µ for the measure of excitability (1/I) increases continuously with elevation of temperature. The form of the F - log I curve does not change except at quite high temperature (35.8°), and then only slightly (near F = 55); F_max. is not altered. The very unusual form of the 1/I curve as a function of temperature is quantitatively accounted for if two processes, with respectively µ = 19,200 and µ = 3,400, contribute independently and simultaneously to the control of the speed of the reaction governing the excitability; the velocities of these two processes are equal at 15.9°.     

Early life-history processes in benthic octopus: Relationships between temperature, feeding, food conversion, and growth in juvenile Octopus pallidus

Initial growth in cephalopods is exponential, making early life-history critical in determining growth trajectories. Few captive studies have however examined the early life-history of cephalopods in fluctuating temperatures as would be encountered in the wild. This study investigates the relationship between early growth and the significant factors affecting growth, namely food intake, food conversion and fluctuating environmental temperatures. Pale octopus (Octopus pallidus) hatchlings were reared in captivity under either a warming or cooling temperature regime. Individual variations and periodicity in feeding rates (F_r), food conversion rates (C_r), growth rates (G_r), and the relationship between these variables and temperature were examined weekly. Food conversion rates were variable between individuals but also within individual octopus, which exhibited large fluctuations in C_r over time, exceeding 100% d^− 1 in one instance. Although individual F_r, C_r and G_r displayed fluctuations over time, there was no evidence of periodicity for any of the variables. Changes in temperature were not significantly correlated to changes in F_r, C_r or G_r. Feeding rate did not appear to influence growth rate or food conversion rate. Food conversion rate was negatively correlated to feeding rate in the same week, and positively correlated to growth rate. Short periods of low or no food consumption were common and the high values obtained for food conversion rate for some individuals suggest that octopus can grow substantially with little or no food intake. Individual variability observed in octopus growth may be dependent on the growth mechanism involved, specifically a fine balance between the continuous hyperplasic and hypertrophic growth found in cephalopods.