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.     

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.     

Energetics of sodium transport in frog skin. I. Oxygen consumption in the short-circuited state

Sodium transport and oxygen consumption were studied simultaneously in the short-circuited frog skin. Sodium transport was evaluated from I _o /F, where I _o is the short-circuit current measured with standard Ringer''s solution bathing each surface and F is the Faraday constant. Oxygen tension was measured polarographically. Under a variety of circumstances the rate of oxygen consumption from the outer solution exceeded that from the inner solution, the ratio being constant (0.57 ± 0.09 SD). Both I _o and the associated rate of oxygen consumption J _ro declined nonlinearly with time, but the relationship between them was linear, suggesting that the basal oxygen consumption was constant. For each skin numerous experimental points were fitted by the best straight line. The intercept (J _ro)_Io=0 then gave the basal oxygen consumption, and the slope dNa/dO₂ gave an apparent stoichiometric ratio for a given skin. The basal oxygen consumption was about one-half the total oxygen consumption in a representative untreated short-circuited skin. Values of dNa/dO₂ in 10 skins varied significantly, ranging from 7.1 to 30.9 (as compared with Zerahn''s and Leaf and Renshaw''s values of about 18). KCN abolished both I _o and J _ro. 2,4-dinitrophenol (DNP) depressed I _o while increasing J _ro four- to fivefold. Anti-diuretic hormone stimulated and ouabain depressed both I _o and J _ro; in both cases apparent stoichiometric ratios were preserved.     

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.     

Oxygen cost of chloride transport in perfused rectal gland ofSqualus acanthias

Summary In the stimulated state, with glucose as substrate, oxygen uptake by the isolated perfused rectal gland is directly related to the rate of chloride secretion. Lactate production is negligible under aerobic conditions in the stimulated gland. A stoichiometric relationship exists between chloride transport and oxygen consumption, with a Cl/O₂ ratio of about 301, resembling that reported for sodium in mammalian kidneys. This ratio remains constant under varying degrees and modes of stimulation. The ratio does not change when the gland is induced to secrete chloride against varying electrochemical gradients by altering the concentration of urea in the perfusate.Established Investigator of the American Heart Association.     

Oxygen consumption by frog skin and its isolated epithelial layers as a function of their sodium-transporting activity

The metabolic cost (in terms of oxygen consumption) of transcellular sodium transport was assessed on ventral frog skin and its isolated epithelial layers, by measuring the decrease in oxygen consumption by the tissue upon transient withdrawal of sodium from the outside solution. The same number of sodium ions was transported per molecule oxygen consumed by whole skin (17.4±2.3) and its isolated epithelium (17.3±2.4).The metabolic cost of sodium transport could not be estimated properly when this process was blocked by amiloride or ouabain, as these drugs were found to bring about an increase in oxygen consumption by the tissue when no sodium was available for transport.     

Oxygen consumption by frog skin and its isolated epithelial layers as a function of their sodium-transporting activity.

The metabolic cost (in terms of oxygen consumption) of transcellular sodium transport was assessed on ventral frog skin and its isolated epithelial layers, by measuring the decrease in oxygen consumption by the tissue upon transient withdrawal of sodium from the outside solution. The same number of sodium ions was transported per molecule oxygen consumed whole skin (17.4 +/- 2.3) and its isolated epithelium (17.3 +/- 2.4). The metabolic cost of sodium transport could not be estimated properly when this process was blocked by amiloride or ouabain, as these drugs were found to bring about an increase in oxygen consumpton by the tissue when no sodium was available for transport.     

Sodium flux ratio through the amiloride-sensitive entry pathway in frog skin

The sodium flux ratio of the amiloride-sensitive Na+ channel in the apical membrane of in vitro Rana catesbeiana skin has been evaluated at different sodium concentrations and membrane potentials in sulfate Ringer solution. Amiloride-sensitive unidirectional influxes and effluxes were determined as the difference between bidirectional 22Na and 24Na fluxes simultaneously measured in the absence and presence of 10(-4) M amiloride in the external bathing solution. Amiloride- sensitive Na+ effluxes were induced by incorporation of cation- selective ionophores (amphotericin B or nystatin) into the normally Na+- impermeable basolateral membrane. Apical membrane potentials (Va) were measured with intracellular microelectrodes. We conclude that since the flux ratio exponent, n', is very close to 1, sodium movement through this channel can be explained by a free-diffusion model in which ions move independently. This result, however, does not necessarily preclude the possibility that this transport channel may contain one or more ion binding sites.     

Interdependence between sodium transport, external chloride, and sodium/calcium exchanger in the isolated skin of the Rana pipiens

The aim of this study was to analyze the relationship of the Na+/Ca2+ exchanger, cytosolic calcium, and chloride to the transepithelial transport of sodium in isolated frog skin. Sodium transport was measured as amiloride-inhibitable short circuit current (SCC). We studied the effect of variations in the concentrations of external chloride and of the manipulation of calcium on sensitive amiloride SCC. Modifications in the movement of Ca2+ were induced by an ionophore, A23187, and a Ca2+ channel blocker, nifedipine. Calcium ionophore A23187 (5 and 20 microM), in a normal Ringer's solution, increased SCC and transepithelial potential difference (PD). In contrast, nifedipine (20 microM) reduced SCC and PD. The role of the Na+/Ca2+ exchanger was studied using dichlorobenzamil (DCB, 50 microM) and quinacrine (1 mM), inhibitors of this exchanger. They selectively increased SCC and PD on the mucosal side of the skin, with no effect on the serosal side. This response occurred only in the presence of extracellular calcium. Replacement of NaCl by sodium methanesulfonate or the addition of furosemide (1 mM) at the serosal compartment, decreased basal SCC and PD and blocked the response to A23187 and the mucosal effect of DCB and quinacrine. These results suggest the presence of an Na+/Ca2+ exchanger located on the mucosal side of the frog skin, which participates in the transepithelial sodium transport. The action of this exchanger may be modulated by external chloride and calcium. J. Exp. Zool. 289:23-32, 2001.     

K Fluxes in Frog Skin

A method has been developed for measuring K influx into the epithelial cells of frog skin from the inside solution. Diffusion delay in the connective tissue has been taken into account. Ninety-four per cent of skin K was found to exchange with K⁴² in the inside solution with a single time constant. K influx showed saturation with increasing K concentration, was not altered by imposing a potential difference of ±200 mv across the skin, and was inhibited by dinitrophenol, fluoroacetate, and ouabain. Relatively low concentrations of dinitrophenol (5 x 10^-5 M) and fluoroacetate (10^-10 M) had no effect on k influx but caused a 40 per cent decrease in net Na flux. There was no correlation between the rate of K uptake at the "inner barrier" and the rate of net Na transport. Reduction of net Na transport by lowering Na concentration in the outside solution caused little change in K uptake. These observations indicate that there is not a significant Na-K exchange involved in active transport of Na across the skin. K influx was found, however, to require Na in the inside bathing solution.     

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.     

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.     

Energetics of coupled active transport of sodium and chloride

A Clark electrode was used to measure oxygen consumption by the gall bladder, in which there is a direct and one-to-one linkage between active Na and active Cl transport. O2 uptake was reversibly depressed when Cl in the mucosal bathing solution was replaced by a poorly transported anion, such as sulfate. This effect of Cl was abolished by ouabain or in Na-free solutions. When the anion was chloride, treatment with ouabain or replacement of Na by a poorly transported cation depressed QO2 more than did replacement of Cl. However, ouabain or removal of Na also depressed QO2 in Na2SO4 solutions, in which salt transport is minimal. It is concluded that oxygen uptake in the gall bladder consists of three fractions: 9% requires both Na and Cl, is inhibited by ouabain, and is linked to the NaCl pump; 36% requires Na but not Cl, is inhibited by ouabain, and possibly is linked to the cellular K uptake mechanism; and 55% represents basal uptake. If the extra oxygen uptake observed during transport supplies all the energy for transport, then 25 Na + 25 Cl ions are transported actively per O2 consumed; i.e., twice as many ions as in epithelia which transport only Na actively. This extra uptake is more than sufficient to supply the energy for overcoming internal membrane resistance under the experimental conditions used.     

The significance of changes in thermodynamic affinity induced by aldosterone in sodium-transporting epithelia

Summary The energetics of sodium transport were examined in toad (and occasionally frog) skin, with particular emphasis on the effect of aldosterone.Thermodynamic affinity was computed according to Essig and Caplan. Following treatment with antidiuretic hormone or drugs believed to affect only the apical membrane barrier, no change in thermodynamic affinity was observed either acutely (after one to two hours) or chronically (after 18-odd hours).By contrast, following treatment with aldosterone overnight, thermodynamic affinity was considerably increased, whether or not incubation was conducted in the presence of sodium in the outer solution; addition of glucose at the end of incubation, whereby sodium transport was stimulated further, failed to influence affinity as measured. The stoichiometry between sodium transport and oxygen consumption was, however, unchanged by aldosterone treatment in short-circuit conditions, neither was that fraction of aerobic metabolism unrelated to sodium transport influenced.It is concluded that the change observed with aldosterone can be directly ascribed to the hormone, as it is independent of glucose availability and of sodium transport. Aldosterone action, at least following prolonged incubation, therefore does not involve only an increase in apical conductance for sodium.     

The antiepileptic drug phenytoin affects sodium transport in toad epithelium

The effects of phenytoin on isolated Pleurodema thaul toad skin were investigated. Low (micromolar) concentrations of the antiepileptic agent applied to the outside surface of the toad epithelium increased the electrical parameters (short-circuit current and potential difference) by over 40%, reflecting stimulation of Na(+) transport, whereas higher (millimolar concentrations, outside and inside surface) decreased both electric parameters, the effect being greater at the inside surface (40% and 80% decrease, respectively). The amiloride test showed that the stimulatory effect was accompanied by an increase and the inhibitory effect by a decrease in the sodium electromotive force (ENa). It is concluded that the drug interaction with membrane lipid bilayers might result in a distortion of the lipid-protein interface contributing to disturbance of Na(+) epithelial channel activity. After applying the Na(+)-K(+)-ATPase blocker ouabain and replacing the Na(+) ions in the outer Ringer's solution by choline, it was concluded that both active and passive transport are involved in sodium absorption, although active transport predominates.     

Transepithelial transport of sodium and chloride ions in isolated skin of the frog,Rana esculenta L

Isolated frog skin, mounted in a Ussing apparatus, was investigated electrophysiologically. Application of amiloride, an inhibitor of sodium ion transport, and bumetanide, known to block the transport of chloride ions, revealed the effect of these ions on PD, both under control conditions and following mechanical stimulation. Under control conditions, mechanical stimulation of the skin caused hyperpolarization, i.e. a transient increase in the electrical potential difference. Preincubation in the presence of amiloride, or amiloride plus bumetanide, brought about both a decrease in electrical potential and an inhibition of the reaction upon stimulation. On the other hand, incubation with bumetanide resulted in a decrease in electrical potential, but did not affect the skin reaction after mechanical stimulation. The above results indicate that hyperpolarization of the frog skin following mechanical stimulation is caused by enhanced transepithelial transport of sodium ions which, in turn, is induced by stimulation of sensory receptors.     

Glutamate transport driven by an electrochemical gradient of sodium ions in Escherichia coli.

The role of Na+ in glutamate transport was studied in Escherichia coli B, strain 29-78, which possesses a very high activity of glutamate transport (L. Frank and I. Hopkins, J. Bacteriol., 1969). Energy-depleted cells were exposed to radioactive glutamate in the presence of a sodium gradient, a membrane potential, or both. One hundred- to 200-fold accumulation of the amino acid was attained in the presence of both electrical and chemical driving forces for the sodium ion. Somewhat lower accumulation values were obtained when either chemical or electrical driving forces were applied separately. A chemical driving force was produced by the addition of external Na+ to Na+-free cells. A membrane potential was established by a diffusion potential either of H+ in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone or of SCN-. These results support the hypothesis of a Na+-glutamate cotransport. Na+-driven glutamate transport was also observed in wild-type E. coli B but not in a strain of K-12.     

Removal of sodium inactivation and block of sodium channels by chloramine-T in crayfish and squid giant axons.

Modification of sodium channels by chloramine-T was examined in voltage clamped internally perfused crayfish and squid giant axons using the double sucrose gap and axial wire technique, respectively. Freshly prepared chloramine-T solution exerted two major actions on sodium channels: (a) an irreversible removal of the fast Na inactivation, and (b) a reversible block of the Na current. Both effects were observed when chloramine-T was applied internally or externally (5-10 mM) to axons. The first effect was studied in crayfish axons. We found that the removal of the fast Na inactivation did not depend on the states of the channel since the channel could be modified by chloramine-T at holding potential (from -80 to -100 mV) or at depolarized potential of -30 mV. After removal of fast Na inactivation, the slow inactivation mechanism was still present, and more channels could undergo slow inactivation. This result indicates that in crayfish axons the transition through the fast inactivated state is not a prerequisite for the slow inactivation to occur. During chloramine-T treatment, a distinct blocking phase occurred, which recovered upon washing out the drug. This second effect of chloramine-T was studied in detail in squid axons. After 24 h, chloramine-T solution lost its ability to remove fast inactivation but retained its blocking action. After removal of the fast Na inactivation, both fresh and aged chloramine-T solutions blocked the Na currents with a similar potency and in a voltage-dependent manner, being more pronounced at lower depolarizing potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Some features of hydrogen (ion) secretion by the frog skin.

We have studied the movements of H+ from the in vitro frog skin into the outside solution because it has been suggested that the movement of sodium from the outside solution into the skin may result from the forced exchange of Na+ by H+. Our main observations can be summarized as follows: (a) Hydrogen moves from the skin into the outside solution at a rate of 0.04 muequiv-cm-2-h-1 while Na+ influx had a value of 0.49 muequiv-cm-2-h-1. (b) The rate of H+ secretion is not significantly affected by substituting the Na+ in the outside solution by K+ nor by inhibiting Na+ influx with amiloride (5-10(-5) M). (c) Acetazolamide (5-10(-3) M) blocked H+ secretion without altering the potential difference across the skin. (d) The rate of H+ production is not underestimated because it may have been neutralized by HCO3- secreted into the outside solution in exchange for Cl-. Substituting all the Cl- by SO4(2-) in the outside solutions does not result in an increase in the rate of H+ production. (e) The steady-state rate of H+ secretion is not affected by large changes in electrochemical potential gradients for H+. Neither abolishing the potential difference across the skin nor a 10-fold change in H+ concentration in the outside solution affected significantly the steady-state rate of H+ secretion. (f) The H+ secretion was abolished by the metabolic inhibitors dinitrophenol (1-10(-4) M) and Antimycin A (1.5-10(-6) M) which also markedly reduced the potential difference across the skin. Observations (a), (b), and (c) suggest that H+ and Na+ movements across the outer border of the isolated frog skin are not coupled. The ratio of Na+ to H+ movements is very different from unity and Na+ movements can be abolished without any effects on H+ secretion and conversely H+ movements can be abolished without interruption of Na+ uptake. A second conclusion suggested by these results is that the H+ secretion does not result from movement of H+ following its electrochemical potential gradient since that rate of secretion is not affected by marked changes in either potential or [H+]. Furthermore, the effects of metabolic inhibitors suggest that H+ secretion requires the expenditure of energy by the cell.     

Effects of lysine-vasopressin (LVP) and 1-deamino-8-D-arginine-vasopressin (dDAVP) upon electrical potential, short-circuit current and transepithelial D.C. resistance of the frog skin

The synthetic analogue of vasopressin, 1-deamino-8-D-arginine-vasopressin (dDAVP), possesses a protracted antidiuretic activity while having practically no pressoric activity as compared to arginine-vasopressin (AVP) or lysine-vasopressin (LVP). The effects of LVP and dDAVP were studied on the frog skin (Rana temporaria) sodium transport as reflected by the short-circuit current (SCC) level, on an Ussing apparatus. The application two different equimolar doses of LVP or dDAVP (approx. 9.4 X 10(-8) mol X l-1 and 18.8 X 10(-8) mol X l-1 to the inner surface of the skin resulted in identical maximal increases of sodium transport. However, the maximum transport stimulation after the application of dDAVP was delayed by about 30 min as compared to the stimulation by LVP (P less than 0.01). In addition, a protracted recovery of SCC towards its original levels was observed in experiments with dDAVP application after the hormone removal (P less than 0.01). It is concluded that dDAVP stimulates Na+ transport through the frog skin despite its lacking pressoric activity. Thus, the natriferic activity of vasopressin is related to its antidiuretic rather than pressoric activity. Maximum increase in the sodium transport following dDAVP application was delayed and more protracted as compared to the effect of LVP.