Voltage-dependent apical conductance in Necturus gallbladder.
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Animals↗
Biomedical subjects
A Essig
Publications and source records attributed to A Essig.
Cellular and paracellular conductance patterns in voltage-clamped frog skin.
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Animals↗
Relationship of transepithelial electrical potential to membrane potentials and conductance ratios in frog skin.
Previous studies in anuran epithelia have shown that, after clamping the transepithelial voltage in symmetrical sequences for 4-6 min there is near-constancy of the rate of active Na transport and the associated oxidative metabolism, with a near-linear potential dependence of both. Here we have investigated in frog skin the cellular electrophysiolgical events associated with voltage clamping (Vt = inside-outside potential). Increase and decrease of Vt produced converse effects, related directly to the magnitude of Vt. Hyperpolarization resulted in prompt decrease in inward transepithelial current It and increase in fractional outer membrane resistance fRo (as evaluated from small transient voltage perturbations) and in outer membrane potential Vo. Overshoot of Vo was followed by relaxation to a quasi-steady state in minutes. Changes in fRo were progressive, with half times of some 1-5 sec. Changes in transepithelial slope conductance gt were more variable, usually preventing precise evaluation of the outer and inner cell membrane conductances go and gi. Nevertheless, it was shown that go is related inversely to Vt and Vo. Presuming insensitivity of gi to Vt, the dependence of Go on Vo in the steady state much exceeds that predicted by the constant field equation. Apparent inconsistencies with earlier results of others may be attributable to differences in protocol and the complex dependence of go on Vo and/or cellular-current. In contrast to previous findings in tight epithelia at open circuit, differences in Vt were associated with substantial differences in fRo and inner membrane potential Vi. Hyperpolarization of Vt over ranges commonly employed in studies of active transport ad metabolism appears to increase significantly the electrochemical work per Na ion transported.
Animals↗
Influence of cellular and paracellular conductance patterns on epithelial transport and metabolism.
Theoretical analysis of transepithelial active Na transport is often based on equivalent electrical circuits comprising discrete parallel active and passive pathways. Recent findings show, however, that Na+ pumps are distributed over the entire basal lateral surface of epithelial cells. This suggests that Na+ that has been actively transported into paracellular channels may to some extent return to the apical (mucosal) bathing solution, depending on the relative conductances of the pathways via the tight junctions and the lateral intercellular spaces. Such circulation, as well as the relative conductance of cellular and paracellular pathways, may have an important influence on the relationships between parameters of transcellular and transepithelial active transport and metabolism. These relationships were examined by equivalent circuit analysis of active Na transport, Na conductance, the electromotive force of Na transport, the "stoichiometry" of transport, and the degree of coupling of transport to metabolism. Although the model is too crude to permit precise quantification, important qualitative differences are predicted between "loose" and "tight" epithelia in the absence and presence of circulation. In contrast, there is no effect on the free energy of metabolic reaction estimated from a linear thermodynamic formalism. Also of interest are implications concerning the experimental evaluation of passive paracellular conductance following abolition of active transport, and the use of the cellular voltage-divider ratio to estimate the relative conductances of apical and basal lateral plasma membranes.
Animals↗
Effects of antidiuretic hormone on kinetic and energetic determinants of active sodium transport in frog skin.
The effects of antidiuretic hormone (ADH) on the rate of transepithelial active Na transport JaNa and the rate of suprabasal O2 consumption of Jsbr were studied in paired hemiskins of frog. Within some 30 min following administration of ADH both JaNa and Jsbr increased to near-maximal levels and then remained stable for at least an hour. On symmetric perturbation of the transepithelial electrical potential delta psi at 6-min intervals, the dependence of JaNa and Jsbr on delta psi was near-linear, both in control and experimental hemi-skins. The stability and near-linearity of the system permitted systematic analysis of the parameters of linear non-equilibrium thermodynamic (NET) and electrical equivalent circuit (EC) formulations. ADH (100 mU/ml) stimulated two of the three NET phenomenological L coefficients, as well as A, the affinity (negative Gibbs free energy) of a metabolic reaction driving transport. Observations at partially depressed levels of transport indicated that the effects of kinetic and energetic factors are to some extent discrete. EC analysis showed stimulation of the amiloride-sensitive conductance Ka, but not of the apparent electromitive force of Na transport 'ENa'. Similar effects were produced by 10 mU/ml of ADH or by 10 mM dibutyryl cyclic AMP, although less marked effects on the L coefficients were noted with the lower concentration of hormone. It is suggested that, in contrast to EC analysis, the NET formulation distinguishes between kinetic and energetic determinants of transport, supporting a dual mechanism of action of ADH.
Animals↗
Time course of active Na transport and oxidative metabolism following transepithelial potential perturbation in toad urinary bladder.
The use of an Ussing chamber with well-defined mixing characteristics coupled to a mass spectrometer permits the concurrent evaluation of transepithelial current and oxidative metabolism with improved temporal resolution. The time-course of the amiloride-sensitive current Ia and the rate of suprabasal CO2 production JsbCO2 were observed in 10 toad urinary bladders at short-circuit and after clamping delta psi at 100 mV, serosa positive. Following perturbation of delta psi (0 leads to 100 mV), Ia declined sharply within 1/2 min, remaining near constant approximately 15 min, and then increased slightly. JsbCO2 declined more gradually, remained near constant at approximately 4-7 min, and then declined further. Detailed analysis revealed an early quasi-steady state with near constancy of JsbCO2 starting at 2.9 +/- 1.1 (SD) min and lasting 4.7 +/- 1.8 (SD) min, followed by relaxation to a later steady state at about 15 min. During the early quasi-steady state, Ia was also nearly constant. Considering that in steady states Ia/F approximately or equal to JaNa, the rate of transepithelial active Na transport, during the early quasi-steady state mean values +/- SE of JaNa, JsbCO2 and (JaNa/JsbCO2) were, respectively, 29.9 +/- 1.7%, 59.4 +/- 3.2%, and 56.4 +/- 5.7% of values at short-circuit. Corresponding values during the late steady state were 41.4 +/- 6.0%, 38.2 +/- 6.1%, and 111.3 +/- 8.6%. Thus the flow ratio JaNa/JsbCO2 was depressed significantly during the early quasi-steady state, but returned later to the original value. The results of measurements of Ia and JsbO2 in three hemibladders were qualitatively similar. In terms of a phenomenological "black-box" treatment the findings are consistent with earlier studies indicating incomplete coupling between transport and metabolism. Further studies will be required to clarify the molecular basis for these observations.
Adenosine Diphosphate↗
Active transport: conditions for linearity and symmetry far from equilibrium.
The impressive linearity of force-flow relationships in epithelial active-transport systems suggests the utility of a linear, nonequilibrium-thermodynamic analysis. We here present a plausibility argument for the appropriateness of such a treatment. Conventional phenomenological equations of nonequilibrium thermodynamics constitute an incomplete description of the processes under study, because a given thermodynamic force may be induced in an infinite number of ways. In general, therefore, flows are nonlinear functions of the forces, and the Onsager reciprocal relations are obeyed only very near equilibrium. If, however, the forces of two coupled processes can be constrained to "proper" pathways, each flow is a linear function of each force, and the phenomenological cross-coefficients are equal far from equilibrium. The nature of such proper pathways is investigated in terms of a simple model of a sodium-active transport system. Where the treatment is appropriate (i.e., for sufficiently small perturbations about a steady state far from equilibrium) it permits a complete thermodynamic characterization of a system, even when only one of the two forces can be controlled experimentally while the other remains constant.
Biological Transport, Active↗
Dynamic aspects of transepithelial active Na transport and oxidative metabolism.
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Animals↗
Protocol-dependence of equivalent circuit parameters of toad urinary bladder.
Determinations o- current-voltage relationships are widely employed in the characterization of epithelial sodium transport. In order to determine the protocol dependence of transport parameters in the toad urinary bladder, studies were carried out in the presence and absence of amiloride, an inhibitor of active sodium transport. With symmetric positive and negative perturbations of the transepithelial electrical potential difference delta psi (0 leads to +/- 100 mV) for 30 sec, the amiloride-sensitive current-voltage (ia-delta psi) relationship was near linear over the range -75 leads to +100 mV, indicating constancy of the conductance ka and the apparent electromotive force "ENa", lumped parameters of the standard electrical equivalent circuit model of the active transport system. With a reverse protocol (+/- 100 leads to 0 mV) or 15 min perturbations the ia-delta psi relationships were highly nonlinear. Nonlinearity reflected voltage dependence of parameters: perturbations that increased active transport decreased "ENa" and increased ka, as evaluated from 10 sec perturbations of delta psi; slowing of active transport produced the converse changes. These effects are usefully analyzed in both quasi-steady states and true steady states by means of a detailed equivalent circuit incorporating the significant ionic currents across each plasma membrane. Precise understanding of the significance of ka and "ENa" will require characterization of the partial ionic conductances on perturbation of delta psi.
Amiloride↗
Nonequilibrium linear behavior of biological systems. Existence of enzyme-mediated multidimensional inflection points.
The linear phenomenological equations of nonequilibrium thermodynamics are limited theoretically to near equilibrium although a number of biological systems have been shown to exhibit a "linear" relationship between steady-state flows and conjugate thermodynamic forces outside the range of equilibrium. We have found a multidimensional inflection point which can exist well outside the range of equilibrium around with enzyme-catalyzed reactions exhibit "linear" behavior between the logarithm of reactant concentrations and enzyme catalyzed flows. A set of sufficient conditions has been derived which can be applied to any enzyme mechanism to determine whether a multidimensional inflection point exists. The conditions do not appear overly restrictive and may be satisfied by a large variety of coupled enzyme reactions. It is thus possible that the linearity observed in some biological systems may be explained in terms of enzyme operating near this multidimensional point.
Enzymes↗
A binding site model of membrane transport: binary and cooperative flows.
The flows of solute molecules in a membrane under the influence of concentration gradients are considered within the framework of classical physical theories. A lattice model is constructed in which the binding sites represent potential minima and the flows are regarded as a result of molecules' making discrete transitions between the binding sites. Expressions for two-component currents are derived from certain descriptions for the transition mechanism. Where the molecular movement is given the crudest description, permeability coefficients are identical for both components and there is no current coupling. Where the molecular movement is given some finer detail, the permeability coefficients differ and positive coupling of flows appears. Our result applies to a combination of flows of tracer and abundant species as well as, more generally, to any combination of flows of two components which are distinguishable yet kinetically similar. Also considered are binary currents whose transport mechanism is further controlled by allosteric cooperativity. Whether the cooperative control is short or long ranged, permeability coefficients and fluxes differ appreciably from those without cooperative control. Thus, unlike in the case of channel flow, current coupling here may be either positive or negative, depending on the strength and nature of cooperative coupling. Numerical evidence suggests that the permeability and coupling may have discontinuous behavior, possibly indicating the existence of phase transitions. Our lattice model, from which the formulations for the flows are obtained, is compatible with current concepts of membrane structure.
Binding Sites↗
Evaluation of the rate of basal oxygen consumption in the isolated frog skin and toad bladder.
In the study of active transport it is important to distinguish between oxygen consumption sustaining transepithelial transport and that responsible for other tissue functions (basal metabolism). Since amiloride blocks transepithelial active sodium transport and the associated oxygen consumption in the frog skin and toad bladder, we and others have employed this agent to evaluate the rate of basal metabolism. This technique has recently been criticized in a report that amiloride (and ouabain) increased oxygen consumption when no sodium was available for transport. We have been unable to corroborate these observations. With magnesium-Ringer as external bathing solutions, amiloride and ouabain failed to stimulate oxygen consumption. With sodium-Ringer as external bathing solution amiloride reduced oxygen consumption about 30%, to a level indistinguishable from that found on external substitution of magnesium-Ringer for sodium-Ringer. We conclude that the use of amiloride permits evaluation of the rate of basal metabolism with acceptable accuracy; a possible slight depressant effect of ouabain on basal metabolism remains to be investigated.
Amiloride↗
Measurement of oxygen consumption in voltage-clamped epithelia.
Standard apparatus for in vitro study of anuran epithelia was adapted for the incorporation of Clark oxygen electrodes, permitting the concurrent measurement of electrical current (active sodium transport) and oxygen consumption under voltage-clamp conditions, with independent manipulation of the two bathing solutions. Stability was enhanced by electrical isolation of the O2 electrodes, sensitive temperature control (+/- 0.03 degrees C), and constancy of stirring rate. Mean rate of drift was less than 3.7% of the average rate of basal (i.e., nontransport related) O2 consumption. In the present configuration the response time was less than 15 s. Visual fitting of slopes of O2 tension evaluated rates of O2 consumption with adequate accuracy in the physiological range (basal rates averaged about 20 pmol-s-1-cm-2 toad bladder, corresponding to about 10 pmol-s-1-mg-1 dry wt, or about 5 pmol-s-1-ml-1 chamber volume). Rpresentative slopes were read with a standard deviation of 3.5%.
Animals↗
The use of linear nonequilibrium thermodynamics in the study of renal physiology.
Classical formulations for the analysis of membrane transport processes, which ignored possible interactions between flows of diverse permeant species, often led to inconsistencies in the evaluation of permeability coefficients. For water flow induced by an osmotic pressure difference this difficulty was resolved by Staverman's introduction of the reflection coefficient sigma, a parameter which incorporates the interaction between solute and solvent in the course of their passage through a membrane. A comprehensive nonequilibrium thermodynamic (NET) formalism suitable for many biological systems was provided by Kedem and Katchalsky. For an n-flow system each flow is in general dependent on n forces; the assumption of Onsager reciprocity, however, reduces the number of independent phenomenological coefficients. Although NET is widely applied in the study of renal physiology, fundamental theoretical and practical problems remain. Basic considerations are the need to control or evaluate the influence of all coupled flows and to establish conditions fostering linear dependencies of flows on forces. When this is done a transport system may be characterized in terms of intrinsic membrane parameters, facilitating the systematic study of the effects of drugs, hormones, and various experimental perturbations.
Biological Transport, Active↗
Use of mass spectrometer to measure CO2 and O2 fluxes in voltage-clamped epithelia.
A quadrupole mass spectrometer was coupled to an Ussing chamber in order to evaluate rates of oxidative metabolism in voltage-clamped epithelia. Well-defined mixing characteristics of the continuously perfused chamber allowed CO2 and O2 concentrations to be related to rates of CO2 efflux, JCO2, and oxygen influx, JO2. The use of a model tissue to simulate step changes in JCO2 validated the treatment, with response within a minute. Monitoring of metabolism was facilitated by use of a desk-top computer, which evaluated JCO2 at 6-s intervals. Concurrent measurements of electrical current and JCO2 were made in the toad urinary bladder in order to relate active sodium transport to metabolism; the use of amiloride to eliminate active transport and the associated metabolism then allowed evaluation of the rates of active Na transport (JNa) and suprabasal metabolism (JsbCO2), and their ratio JNa/JsbCO2. We report the ability to resolve a 5 pmol/s change in CO2 efflux or an 11 pmol/s change in O2 influx rates.
Animals↗
Evaluation of kinetic and energetic parameters of active sodium transport.
Active sodium transport is classically analyzed in terms of an equivalent circuit, comprising an active conductance Ka and an electromotive force of sodium transport ENa. Although ENa is commonly considered the driving force of transport, model experiments have suggested that ENa is a composite parameter, incorporating both kinetic and energetic factors. An alternative approach considers both transport and the associated oxidative metabolism in terms of a nonequilibrium thermodynamic (NET) formulation, involving phenomenological coefficients and the affinity A, presumed to represent kinetic and energetic factors, respectively. Model experiments testing the NET formulation suggest that the affinity is indeed an energetic parameter. Calculated values of A in untreated frog skins and toad bladders range from about 20 to 80 kcal per mole of O2 consumption. Assuming a P/O ratio of 3, this range corresponds to about 3--13 kcal per mole of ATP utilization, values compatible with reported direct measurements. Although brief perturbations of transepithelial electrical potential deltapsi resulted in linear current-voltage relationship, indicating constancy of ENa and Ka, 15-min perturbations of deltapsi resulted in nonlinearity, indicating changes in ENa and Ka; perturbations of deltapsi enhancing active transport were associated with decrease of ENa and increase of Ka; slowing of active transport produced the converse effects.
Animals↗
Kinetics of bidirectional active sodium fluxes in the toad bladder.
The kinetics of isotopic Na+ flows was studied in urinary bladders of toads from the Dominican Republic. Initial studies of the potential dependence of passive serosal to mucosal 22Na+ efflux demonstrated the absence of isotope interaction and/or other coupling with passive Na+ flow. The electrical current I and mucosal to serosal 22Na+ influx were then measured with transmembrane potential clamped at deltapsi=0, 25, 50, 75 or 100 mV. Subsequent elimination of active Na+ transport with mucosal amiloride permitted calculation of the rates of active Na+ transport JaNa and active and passive influx leads to JaNa and leads to JpNa. The results indicate that for Dominican toad bladders mounted in chambers only Na+ contributes significantly to transepithelial active ion transport; hence JaNa=Ja. Ja was abolished at deltapsi=E=96.3+/-1.9 (S.E.) mV. As deltapsi approached E, active efflux comes from Ja became demonstrable. At deltapsi=100 mV, comes from Ja exceeded leads to Ja, so that Ja was negative. Experimental values of leads to Ja agreed well with theoretical values predicted by a thermodynamic formulation: leads to Jaexp=0.985 leads to Jatheor (r=0.993). The dependence of leads to Ja on deltapsi is curvilinear.
Amiloride↗
Ion tracer flows and flux ratios in heterogeneous membranes.
Ion tracer flows and flux ratios at various electrical potentials were studied in heterogeneous membranes comprising parallel pathways of different intrinsic resistance. The total resistance to net flow exceeded the tracer exchange resistance, and the flux ratio was abnormal, as in exchange diffusion commonly attributed to a mobile carrier.
Biophysics↗