William A. Brodsky

The accelerating effect of serosal HCO3- on Na+ transport in short-circuited turtle bladders.

Abstract The isohydric addition of HCO3− to the serosal fluid (final concn., 17 mM) of short-circuited turtle bladders produces a sustained increase (near-doubling) in the rate of Na+ transport from mucosa to serosa. A qualitatively similar, but smaller, effect was obtained from addition of Cl− to the serosal fluid. The anion-induced acceleration of Na+ transport was independent of the presence or absence of HCO3− and/or Cl− in the mucosal fluid. Whereas the transport-accelerating effect of HCO3− could be superimposed upon that of Cl−, the reverse did not hold. The presence and utilization of glucose is apparently an absolute requirement for eliciting the HCO3− effect on Na+ transport. On the other hand, the effect was elicited in anoxic as well as in oxygenated bladders. The O2-induced recovery of Na+ transport from N2-anoxia is dependent upon the presence of serosal HCO3−.

The Means of Distinguishing between Hydrogen Secretion and Bicarbonate Reabsorption: Theory and Applications to the Reptilian Bladder and Mammalian Kidney

Publisher Summary When the concentration of luminal HCO 3 remains fixed or increases during reabsorption, factors such as bulk flow and sieving complicate the interpretation of methods currently used to identify the acidification mechanism. This chapter discusses the theoretical and experimental methods used to distinguish one type of acidification mechanism from another—first, in a general sense, second, in the particular case of the reptilian bladder, and third, in the particular case of the mammalian renal tubule. The weight of available evidence supports the concept that an active HCO 3 transport is the mechanism of luminal acidification in the turtle bladder. This mechanism is the unique explanation for the data on decreasing luminal [CO 2 ] during acidification and is a fully adequate explanation for increasing luminal [CO 2 ] during acidification, H-accumulation in a luminal fluid initially devoid of HCO 3 and CO 2, and the HCO 3 -sensitive short-circuiting current.

The (Na+ + K+)-dependent ATPase in the isolated mucosal cells of turtle bladder

1. 1. In the microsomal fraction of isolated mucosal cells of turtle bladders, addition of Na+ and K+ to Mg2+-containing mixtures increased ATPase activity by 100–150%; and the (Na+ + K+)-dependent activity was completely inhibited by ouabain. 2. 2. Apparent Km values, with respect to ATP were: 0.22 mM for the (Na+ + K+)-dependent and 0.18 mM for the Mg+-dependent activity. The apparent Km for Na+ was 4.5 mM; and that for K+, 0.36 and 2.9 mM. 3. 3. No effects on activity were found after addition of amiloride, furosemide, and acetazolamide. In contrast, 0.1 mM N-ethylmaleimide inhibited the (Na+ + K+)-dependent activity by 45% without affecting the Mg2+-dependent activity. 4. 4. For Mg2+-dependent ATPase, the order of nucleotide preference was: ATP > GTP > ITP > CTP > UTP > ADP; and for (Na+ + K+)-dependent ATPase, the order was: ATP > ITP > CTP > GTP, while the activity with UTP or ADP was negligible. 5. 5. The pH optimum of the activity with Na+ + K+ + Mg2+ was 7.3–7.5; while that with Mg2+ alone was a weak function of the pH. 6. 6. Activity of ATPase increased as a function of temperature (15–45°). The calculated activation energy was 13.0 kcal/mole for the Mg2+-dependent and 27.5 kcal/mole for the (Na+ + K+)-dependent activity.

Functions of the E-ATP and E-P complexes in the membrane ATPase reaction

Abstract 1. 1. In turtle bladder microsomes incubated with 0.01 mM ATP at 0°, the Mg-dependent ATPase is 50 % greater than the Ca-dependent ATPase. The addition of Na + + K + induces a 20 % increase of Mg-ATPase, but has no effect on Ca-ATPase. The addition of Ca 2+ to the (Mg 2+ + Na + + K + )-containing system eliminates the (Na + + K + ) increment of its ATPase activity. 2. 2. The E -ATP formed after incubation of microsomes with [U- 14 C]ATP (0.01 mM ATP at 0°) amounts to an average value of 13 pmoles/mg in the presence of Ca 2+ alone or of Mg 2+ alone. The addition of either Na + or K + does not affect the underlying E -ATP formation in the presence of Mg 2+ and/or Ca 2+ . However, the addition of Na + and K + together induces a 32 % increase in the Ca-dependent formation of E -ATP, but no change in the Mg-dependent formation. The addition of Ca 2+ to the (Mg 2+ + Na + + K + )-containing system causes little or no change in ATP binding. 3. 3. The phospho-protein ( E - P ) formed after incubation of microsomes with [γ- 32 P]ATP (0.01 mM ATP at 0°) amounts to an average value of 19.2 pmoles/mg in the presence of Ca 2+ alone, and 76 in the presence of Mg 2+ alone. The addition of Na + causes a 3-fold increase in the amount of Ca-dependent formation of E - P , and a 2-fold increase in the Mg-dependent formation. The addition of K + to the (Ca 2+ + Na + )-containing mixture reduces the level of E - P to the underlying Ca-dependent level; while the same K addition to the (Mg 2+ + Na + )-system reduces the level of E - P formation to less than half (31 pmoles) of the underlying Mg-dependent level. The addition of Ca 2+ to either the ((Mg 2+ + Na + ) or the (Mg 2+ + Na + + K + ) system produces no change in phospho-protein formation. 4. 4. Present data provide support for the hypothesis holding that the presence of Mg 2+ , Na + and K + together is required in the first intermediary reaction step of the (Na + + K + )-ATPase sequence. Although Ca can substitute for Mg in the first step of the (Na + + K + )-ATPase sequence, it apparently forms a stable (Ca 2+ + Na + + K + )- E -ATP complex which does not break down into the final hydrolytic products. It is inferred that the Na-induced increment of phosphoprotein formation occurs in a reaction path which is in parallel to that of (Na + + K + )-ATPase. Possible roles of E -ATP and E - P in sodium transport are adduced.

Identification of intact ATP bound to (Na+ + K+)-ATPase

Abstract 1. 1.|Native and ouabain-treated microsomes of turtle bladder epithelial cells incubated at 0° with [U-14C]ATP form a Mg2+-dependent, acid-stable complex with 14C in a cooperative homotropic manner. 2. 2.|The bound 14C is readily identifiable as intact ATP by first cleaving the 14C from the 14C-labeled microsomal precipitate and by the subsequent chromatographic recovery of 14C-labeled ATP in the supernatant fluid. 3. 3.|The formation of E-ATP is ouabain inhibitable only in the presence of Mg2+ + Na+ + K+; and conversely the formation of E-ATP in the ouabain-treated enzyme in the presence of Mg2+ is inhibitable by addition of Na+ and K+ together. This suggests that at least part of the E-ATP complex is an integral part of the (Na+ + K+)-ATPase system. 4. 4.|The bond between the enzyme and ATP, probably a covalent one, resists increases in ionic strength and osmolality as well as increases in hydrogen bond dissolution. 5. 5.|The stoichiometric relations and turnover numbers of E-ATP and phosphoproteins are estimated with respect to their relative contributions to the overall catalyzed rate of hydrolysis of ATP.

Binding of ATP to and release from microsomal (Na+ + K+)-ATPase.

Abstract 1. 1. Turtle bladder microsomes, which possess (Na+ + K+)-ATPase activity, form Mg2+-dependent acid-stable, radioactively labelled complexes during their incubation at pH 7.4 with 0.01 mM concentrations of [U-14C]ATP, [γ-32P]ATP, and [α-32P]ATP. 2. 2. The formation of a 14C-labelled enzyme-ATP complex is demonstrated by the chromatograhic isolation of intact [U-14C]ATP which had been cleaved (by 3 different chemical methods) from its binding sites on the 14C-labelled microsomal precipitates; and this is confirmed by an analogous isolation of intact [γ-32P]ATP cleaved from γ-32P-labelled precipitates. 3. 3. The pH-dependent patterns of the formation of native complexes and of the breakdown of the acid-denatured complexes show that the 14C-labelled microsomal protein is different from the Na+-dependent γ-32P-labelled protein. The pH dependency of 14C labelling of native microsomes incubated with [U-14C]ATP is consistent with the behavior expected of a phosphoramido bond; while that of γ-32P labelling of native microsomes incubated with [γ-32P]ATP is consistent with the behavior expected of an acyl phosphate bond. 4. 4. The enzyme-ATP complex (or complexes) possesses at least two bonds between the enzyme and ATP: one between the γ-phosphate of ATP and the protein; and the other between the adenosine of ATP and the protein. The presence of an adenosinyl-protein bond is suggested by the fact that the same reagent removes all of the α-32P label but only half of the U-14C label from paired sets of microsomal complexes incubated under identical conditions except for the radioactive label (α-32P vs U-14C) on the substrate (ATP). 5. 5. The pH-dependent pattern of 14C labelling of microsomes by [U-14C]ATP is nearly superimposable upon that of (Na+ + K+)-ATPase activity, while that of the Na+-stimulated γ-32P labelling of microsomes by [γ-32P]ATP is nearly superimposible upon the mucosal pH dependence of Na+ transport in the intact tissue. 6. 6. The addition of 1.0 mM ATP or ADP chases half of the 14C label from native microsomes which had become labelled during their incubation with 0.01 mM [U-14C]ATP. The significance of these and other data with respect to the reversibility of the formation of an enzyme-ATP complex and its role in ADP:ATP exchange is discussed.

Carrier-Mediated Transport Processes

In Chapter 20(1) the movement of solutes across biological membranes is treated in relation to processes which do not, in general, involve a chemical interaction between the permeant and the membrane, and which are thermo-dynamically dissipative in character—i.e., the free energy of the matter under observation decreases during transport.

The effect of temperature on Mg2+− and (Na+ + K+)-ATPases

1. 1. As the incubation temperature is increased from 38 to 80°, the ATPase activity of native and/or ouabain-treated microsomes of turtle bladder epithelial cells increases, reaches maximal levels, and decreases. 2. 2. The maximal activity occurs at 60–63° in native or ouabain-treated microsomes incubated in the presence of Mg2+ alone, Mg2+ + Na+ Mg2+ + K+. The maximal activity occurs at 50° in native microsomes incubated in the presence of Mg2+ + Na+ + K+; and at 58° in ouabain-treated microsomes in the presence of Mg2− + Na+ + K+. 3. 3. The addition of ouabain stimulates ATPase activity at temperatures above 38° in the presence of Mg2+, or in the presence of Mg2+ + Na+, or in the presence of Mg2+ + K+, but not in the presence of all three. This stimulatory action of ouabain is elicited in aging diluted suspensions, not in freshly extracted, concentrated suspensions of microsomes. 4. 4. Between 38 and 50°, the addition of ouabain partially inhibits the ATPase activity in the presence ofMg2+ + Na + + K+ (presumably by suppression of the Na+ + K+ increment of the total activity). Between 55 and 80°, oubain has no apparent effect on ATPase activity in the presence of Mg2+ + Na+ + K+. 5. 5. The addition of either Na+ or K+ decreases the Mg2+-dependent activity of the native and ouabain-treated microsomes at temperatures above 38°. This inhibition is independent of the age or degree of dilution of the microsomal suspension. A qualitatively similar inhibition after the simultaneous addition of Na+ and K+ is elicited above 55°. 6. 6. From 38 to 50°, the simultaneous addition of Na+ and K+ doubles the Mg2+- dependent activity in native microsomes. 7. 7. ATPase activity in the presence of Mg2+ alone was significantly greater than that in the presence of Mg2+ + Na+ + K+ + ouabain over a wide range of temperatures. 8. 8. The free energy of activation, E∗, and the free energy, δF∗, of the ATPase-catalyzed hydrolysis were estimated. 9. 9. An attempt was made to correlate the effects of Na+, K+, and ouabain on some of the properties of ATPase with hydrogen bonding.

Some properties of the ADP-ATP exchange reaction in turtle bladder microsomes

Abstract The catalyzed exchange reaction, [ 14 C]ADP + ATP ⇌ [ 14 C]ATP + ADP , found in the microsomal pellet from isolated mucosal epithelial cells of the bladder of fresh water turtles has an absolute requirement for Mg2+ in the presence or absence of Na+ and/or of Na+ + K+. The rate of exchange is reduced by Na+ and is increased by ouabain over a wide range of Mg2+ levels. The Na+-induced decrement of exchange is elicited in the presence and in the absence of ouabain. In fact, Na+ can entirely abolish the exchange reaction in the native microsomal proteins. The rate of exchange in the presence or absence of Na+ is independent of the pH over a wide pH range. Whereas oligomycin inhibited hydrolysis without affecting the exchange rate, N-ethylmaleimide inhibits both processes. The nucleotide preference of the exchange rate with Mg2+ alone was ATP > GTP > ITP > UTP > CTP; and with Mg2+ + Na+ was ATP > CTP > ITP > UTP > GTP.

Amiloride-induced stimulation of HCO3− reabsorption in turtle bladder

Amiloride in the mucosal fluid (at concentrations of 5 . 10(-6) M to 10(-4) M) reversibly stimulates the HCO-3-dependent moiety of the short-circuiting current (Isc) in ouabain-treated turtle bladders bathed by Na-free Ringer solutions with or without Cl-. This effect is uniquely different from the known inhibitory effect of this agent on Na+ transport. Thus, any comprehensive hypothesis on the action of amiloride over a wide dosage-response range should take into account its effect on HCO-3 transport.