Insulin-stimulated sodium transport in toad urinary bladder

Mammalian and teleost insulins increase active sodium transport by the toad urinary bladder at subnanomolar concentrations. This stimulation is evident within 15 min and persists for hours. Porcine proinsulin and a cross-linked derivative of bovine insulin are less effective than porcine insulin in stimulating the short-circuit current (SCC), indicating the specificity appropriate for activation of sodium transport through an insulin receptor. The initial stimulation by insulin of the SCC is not blocked by pretreatment with actinomycin D, puromycin, cycloheximide, or tunicamycin. However, in the presence of any one of these inhibitors the sustained increase in SCC is blocked and the rise is short-lived, lasting only 45 to 90 min. In amphotericin-treated bladders, the addition of insulin did not further stimulate SCC.     

Effect of sodium transport inhibition on active phosphate transport by toad bladder

Summary The relationship between active Na transport (estimated by the short-circuit (SCC)) and active inorganic phosphate (P_i) transport was studied in the toad bladder. When SCC was inhibited by amiloride, ouabaim, or removal of K from the serosal bathing solution, active P_i transport was totally inhibited. When Na was replaced isotonically by choline in either the mucosal bathing solution or both the mucosal and serosal bathing solutions, there was no measurable SCC or active P_i transport. These experiments are compatible with the hypothesis that active P_i transport occurs only in the presence of active Na transport.     

Metabolic cost of sodium transport in toad urinary bladder

Summary The metabolic cost of active sodium transport was determined in toad bladder at different gradients of transepithelial potential, , 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,J _Na, and suprabasal CO₂ production, , were calculated. Since large transients inJ _Na and frequently accompanied any abrupt change in , steady state conditions were carefully defined.Some 20 to 40 min were required after a change in 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 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 ofJ _Na nor, consequently, of .The independence of the ratio from over the range ±50 mV indicates a high degree of coupling between active sodium transport and metabolism.     

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.     

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.     

Active sodium transport by the isolated toad bladder

Studies were made of the active ion transport by the isolated urinary bladder of the European toad, Bufo bufo, and the large American toad, Bufo marinus. The urinary bladder of the toad is a thin membrane consisting of a single layer of mucosal cells supported on a small amount of connective tissue. The bladder exhibits a characteristic transmembrane potential with the serosal surface electrically positive to the mucosal surface. Active sodium transport was demonstrated by the isolated bladder under both aerobic and anaerobic conditions. Aerobically the mean net sodium flux across the bladder wall measured with radioactive isotopes, Na²⁴ and Na²², just equalled the simultaneous short-circuit current in 42 periods each of 1 hour's duration. The electrical phenomenon exhibited by the isolated membrane was thus quantitatively accounted for solely by active transport of sodium. Anaerobically the mean net sodium flux was found to be slightly less than the short-circuit current in 21 periods of observation. The cause of this discrepancy is not known. The short-circuit current of the isolated toad bladder was regularly stimulated with pure oxytocin and vasopressin when applied to the serosal surface under aerobic and anaerobic conditions. Adrenaline failed to stimulate the short-circuit current of the toad bladder.     

Amide transport channels across toad urinary bladder

Summary Urea and other small amides cross the toad urinary bladder by a vasopressinsensitive pathway which is independent of somotic water flow. Amide transport has characteristics of facilitated transport: saturation, mutual inhibition between amides, and selective depression by agents such as phloretin. The present studies were designed to distinguish among several types of transport including (1) movement thought a fixed selective membrane channel and (2) movement via a mobile carrier. The former wold be characterized by co-transport (acceleration of labele amide flow in the direction of net flow in the opposite direction). Mucosal to serosal (MS) and serosal to mucosal (SM) permeabilities of labeled amides were determined in paired bladers. Unlabeled methylurea, a particularly potent inhibitor of amide movement, was added to either the M or S bath, while osmotic water flow was eliminated by addition of ethylene glycol to the opposite bat. Co-transport of labeled methylurea and, to a lesser degree, acetamide and urea with unlabeled methylurea was observed. Co-transport of the nonamides ethylene glycol and ethanol could not be demonstrated. Methylurea did not alter water permeability or transmembrane electrical resistance. The demonstration of co-transport is consistent with the presence of ADH-sensitive amide-selective channcels rather than a mobile carrier.     

Barium inhibition of sodium ion transport in toad bladder

The effect of Ba²⁺ on Na⁺ transport and electrical characteristics of toad bladder was determined from change produced in short circuit current (I_sc, epithelial, apical and basal-lateral potentials (ψ_t, ψ_a, ψ_b), epithelial and membrane resistances (R_t, R_a, R_b) and shunt resistance (R_s). Mucosal Ba²⁺ had no effect. Serosal Ba²⁺ reduced I_sc, ψ_t, ψ_a, and ψ_b, but had no effect on R_t, R_a, R_b and R_s. Minimal effective Ba²⁺ concentration was 5 · 10^?5 M. The phenomenon was reversed by Ba²⁺ removal, but not by 86 mM serosal K⁺. Ba²⁺ inhibition of I_sc did not impair the response to vasopressin which was quantitatively the same as controls. ψ_a with Ba²⁺ equalled ψ_b. After Ba²⁺ inhibition, ouabain produced no further decrease in ψ_t and I_sc. Ba²⁺ exposure after ouabain did not decrease ψ_t and I_sc. The results suggest that Ba²⁺ inhibits the basal-lateral electrogenic Na⁺ pump.     

Current-voltage analysis of apical sodium transport in toad urinary bladder: Effects of inhibitors of transport and metabolism

Summary The basal-lateral surface of the epithelium of the urinary bladder of the toad (Bufo marinus) was depolarized by exposure of the serosal surface to 85mm KCL and 50mm sucrose. The extent of depolarization appeared to be virtually complete, as evaluated by the invariance in the transepithelial electrical potential difference and conductance on addition of nystatin (a monovalent cation ionophore) to the serosal medium. The Na-specific current (I _Na) was defined as the current sensitive to the removal of Na from the mucosal medium or inhibitable by addition of amiloride to this medium. In the presence of the high K-sucrose serosal medium, rapid, serial, stepwise clamping of the transepithelial voltage (V) yielded a curvilinear dependence ofI _Na onV; which is taken to represent theI–V curve of the apical Na channels. The constant field equation (Goldman, D.E. 1943;J. Gen. Physiol. 27:37) fits theI–V data points closely, allowing estimates to be made of the permeability to Na of the apical membrane (P _Na) and of the intracellular Na activity (Na _c ). Exposure of the apical surface to amiloride (5×10^–7 m) decreasedP _Na in proportion to the decrease inI _Na (i.e., 70%) but decreased Na _c only 25%. In contrast, an equivalent lent reduction inI _Na elicited by exposure of the basallateral surface to ouabain was accompanied by only a 20% decrease inP _Na and a sixfold increase in Na _c . The effects of amiloride onP _Na and ouabain on Na _c are consistent with the primary pharmacological actions of these drugs. In addition,P _Na appears to be under metabolic control, in that 2-deoxyglucose, a specific inhibitor of glycolysis, decreasedI _Na andP _Na proportionately, and lowered Na _c marginally, effects indistinguishable from those obtained with amiloride.     

Action of ouabain on sodium transport in toad urinary bladder, Evidence for two pathways for sodium entry

The cardiac glycoside ouabain inhibits transepithelial sodium transport in the toad urinary bladder. It is shown that this drug reduces the rate coefficient for sodium exit at the serosal pump site. In addition, ouabain inhibits entry across the mucosal border whenever the electrochemical potential gradient for sodium is made less favorable. The data are interpreted as indicating the existence of two separate pathways for sodium entry, one of which is ouabain inhibitable.