Philip R. Steinmetz

Characteristics of stimulation of H+ transport by aldosterone in turtle urinary bladder.

Aldosterone stimulates not only Na+ absorption but also urinary acidification. In this investigation the effects of aldosterone on H+ transport are examined in vitro in turtle bladder, a urinary membrane in which several of the factors controlling H+ transport have been defined. H+ transport was increased in bladder halves exposed to aldosterone compared to control halves. Stimulation of H+ secretion was observed as early as 1 h after addition of aldosterone and occurred before that of Na+ transport. In bladders depleted of endogenous substrate addition of glucose increased H+ transport more in aldosterone-treated halves (10.0+/-1.3 nmol/min) than in control halves (6.8+/-2.3). Addition of pyruvate failed to increase H+ transport (--0.3+/-0.7) in control halves but caused significant increments (2.4+/-0.5) in aldosterone-treated halves. In aldosterone-treated bladders glucose caused larger increments (16.5+/-2.7) in H+ transport than pyruvate (9.3+/-2.0) when halves of the same bladders were compared. Na+ transport, however, was equally increased by the two substrates. Despite the differences in time course and substrate requirements between the stimulation of H+ and Na+ transport, both increases were abolished by actinomycin-D. To examine the effect of aldosterone on the force of the H+ pump, protonmotive force, the pH gradient that would nullify the transport rate was determined with and without aldosterone. Aldosterone did not alter protonmotive force but significantly increased the slope of the H+ transport rate on the applied pH gradient. It is concluded that aldosterone stimulates H+ transport independently of Na+ transport. It increases the responsiveness of the transport rate to glucose and to a lesser extent pyruvate, an effect probably secondary to the increased transport rate. Equivalent circuit analysis indicates that aldosterone facilitates the flow of protons through the active transport pathway but does not increase the force of the pump.

Inhibition of the Bicarbonate Exit Step in Urinary Acidification by a Disulfonic Stilbene

Acidification of the luminal solution by the isolated turtle bladder involves H(+) secretion by a pump at the luminal membrane. The OH(-) dissociated in this process reacts with CO(2) and forms HCO(3) (-) which moves passively out of the cell across the serosal cell membrane. In the present study, this exit step for HCO(3) (-) was inhibited by serosal addition of the disulfonic stilbene, SITS, an agent which is thought to bind to a transport protein at the serosal cell membrane. 90 min after serosal addition of 0.5 mM SITS, H(+) secretion decreased by > 80%. In contrast, luminal addition of SITS had no effect. During inhibition of H(+) secretion by serosal SITS, overall cell pH, measured by the 5, 5-dimethyl-2, 3-oxazolidinedione method, increased from 7.48+/-0.03 to 7.61+/-0.02. This increase of 0.13+/-0.02 pH U was associated with a much larger regional pH increase as judged from the decrement in the attainable pH gradient across the epithelium. After serosal SITS, this gradient was reduced from 2.88+/-0.06 to 2.09+/-0.11 pH U. In the absence of evidence for increased H(+) permeability or a change in the force of the H(+) pump, the gradient decrement of 0.79+/-0.08 U reflects a similar pH increment on the cytoplasmic side of the pump.SITS inhibits the exit of bicarbonate across the serosal cell membrane and, thereby, creates a compartment of high alkalinity in series with the pump. The increased electrochemical gradient across the active transport pathway is the primary factor in the inhibition of urinary acidification.

Coupling between H+ transport and anaerobic glycolysis in turtle urinary bladder: effects of inhibitors of H+ ATPase

SummaryThe coupling between H+ transport (JH) and anaerobic glycolysis was examinedin vitro in an anaerobic preparation of turtle urinary bladder.JH was measured as the short-circuit current after Na+ transport was abolished with ouabain and by pH stat titration. The media were gassed with N2 and 1% CO2 (PO2<0.5 mm Hg) and contained 10mm glucose. Under these conditions,JH was not inhibited by 3mm serosal (S) cyanide or by 0.1mm mucosal (M) dinitrophenol. Control anerobic lactate production (Jlac) of 47 bladders was plotted as a function of simultaneously measuredJH. The slope ofJlac onJH was 0.58±0.12 with an intercept forJlac atJH=0 of 0.55 μmol/hr. Values for δJlac/δJH were determined in groups of individual bladders whenJH was inhibited by an opposing pH gradient (0.55±0.16), by acetazolamide (0.58±0.19) and by dicyclohexylcarbodiimide, DCCD (0.58±0.14). The constancy of δJlac/δJH indicates a high degree of coupling betweenJH andJlac. Since the anaerobic metabolism of glucose produces one ATP for each lactate formed, the δJlac/δJH values can be used to estimate the stoichiometry of H+ translocation. The movement of slightly less than 2 H+ ions is coupled to the hydrolysis of one ATP. During anaerobiosis (absence of mitochondrial ATPase function) the acidification pump was not inhibited byM addition of oligomycin but was inhibited byM addition of DCCD and Dio-9, inhibitors of H+ flow in the proteolipid portion of H+-translocating ATPases. DCCD inhibited anaerobicJH without change in δJlac/δJH or basalJlac and, therefore, acted primarily on the H+ pump.S addition of vanadate also inhibitedJH, but the inhibition was associated with an increase inJlac. The site of this apparent uncoupling remains to be defined. The acidification pump of the luminal cell membrane of the turtle bladder has H+-ATPase characteristics that differ from mitochondrial ATPase in that H+ transport is oligomycin-resistant and vanadate-sensitive. As judged from the flows of H+ and lactate, the H+/ATP stoichiometry of the pump is about 2.

Pathways for bicarbonate transfer across the serosal membrane of turtle urinary bladder: Studies with a disulfonic stilbene

SummaryBicarbonate is transferred across the serosal (S) membrane of the epithelial cells of the turtle bladder in two directions. Cellular HCO3− generated behind the H+ pump moves across this membrane into the serosal solution. This efflux of HCO3− is inhibited by SITS (4-isothiocyano-4′-acetamido-2,2′-disulfonic stilbene). When HCO3− is added to the serosal solution it is transported across the epithelium in exchange for absorbed Cl−. This secretory HCO3− flow traverses the serosal cell membrane in the opposite direction. In this study the effects of serosal addition of 5×10−4m SITS on HCO3− secretion and Cl− absorption were examined. The rate of H+ secretion was brought to zero by an opposing pH gradient, and 20mm HCO3− was added toS. HCO3− secretion, measured by pH stat titration, was equivalent to the increase inM→S Cl− flux after HCO3− addition. Neither theS→M flux of HCO3− nor theM→S flux of Cl− were affected by SITS. In the absence of electrochemical gradients, net Cl− absorption was observed only in the presence of HCO3− in the media; under such conditions, unidirectional and net fluxes of Cl− were not altered by serosal or mucosal SITS. H+ secretion, however, measured simultaneously as the short-circuit current in ouabain-treated bladders decreased markedly after serosal SITS. The inhibition of the efflux of HCO3− in series with the H+ pump and the failure of SITS to affect HCO3− secretion and Cl− absorption suggest that the epithelium contains at least two types of transport systems for bicarbonate in the serosal membrane.

Nephrology rounds, University of Iowa Hospitals: renal tubular acidosis.

We have discussed two patients who had renal tubular acidosis complicated by hypokalemia. The first patient had a distal acidifying defect. Circumstantial evidence has been presented suggesting that exposure to toluene-diisocyanate or toluene-diamine played a role in the pathogenesis. The acidosis and the hypokalemia of this patient were easily corrected by the administration of small amounts of sodium bicarbonate without potassium supplementation. The second patient had an interstitial nephritis of unknown etiology and presented with moderate renal insufficiency, renal tubular acidosis, and proximal as well as distal acidifying defects. The proximal tubular dysfunction was associated with general aminoaciduria and glucosuria. This patient required large quantities of both alkali and potassium to correct the electrolyte abnormalities. The mechanisms of potassium wasting in proximal and distal renal tubular acidosis are reviewed. A classification is presented of cellular defects that may underlie the different renal acidifying defects. Attempts to distinguish between pump and permeability defects from urinary pCO2 levels must take into account the simultaneous HCO-3 concentration, since large pCO2 elevations require the presence of ample HCO-3 in the urine. Permeability defects may impair urinary acidification by either abnormal back flux of H+ out of the lumen or increased influx of HCO-3 into the lumen. In studies of acidification in vitro, amphotericin B causes increased H+ permeability and has little effect on HCO-3 permeability. Toluene-diamine causes a marked permeability defect which is reversible, but remains to be defined in terms of the ion species, HCO-3 or H+, affected. At times, hyperchloremic acidosis is caused by distal defects in net acid excretion that occur without impairment of the H+ gradient. In certain patients with hypoaldosteronism, for example, distal H+ secretion may be reduced without change in the force of the H+ pump.

FUNCTIONAL ORGANIZATION OF PROTON AND BICARBONATE TRANSPORT IN TURTLE URINARY BLADDER

The bladder of the fresh water turtle, Pseudemys scripta, is a relatively “tight” epithelium that resembles amphibian urinary bladders and frogskin in its capacity to absorb sodium by active transport. The major advantage of the turtle bladder preparation is it contains a potent transport system for urinary acidification that lends itself to close examination under simplified and controlled conditions in vitro. Substantial evidence is now available to suggest that the energy-dependent step in acidification is located at the luminal cell membrane and involves the translocation of protons from the cell into the urinary compartment.2-5 The nature of the acidification pump remains to be defined precisely, but recent evidence B. 7 points toward a proton-translocating ATPase that shares some of the characteristics of H+-ATPases in mitochondria, chloroplasts and bacterial membranes. Since net transport across epithelia requires translocation of ions across two cell membranes in series, the organization of the acidification pathway is more complex than in single membrane systems. As shown in FIGURE 1, the extrusion of H+ ions across the luminal membrane results in an accumulation of OHions within the cell. In the presence of CO? and carbonic anhydrase, HC0,will be generated that moves “downhill” across the basolateral cell membrane into the serosal This acidification pathway was first defined in a system free of exogenous CO, and HC0,in which the production and distribution of metabolic CO, and HC0,were measured by means of a conductometric t echn iq~e .~ . Studies by Steinmetz, Omachi and Frazier and Schwartz and Steinmetz s demonstrated that in this system H+ transport is electrogenic and not coupled directly to the transport of sodium. Accordingly in FIGURE I , the two transport systems are shown separately. When net sodium absorption is abolished by addition of ouabain or removal of sodium the electrical potential difference across the tissue reverses so that the lumen becomes positive with respect to the serosa and the reversed short-circuit current becomes equal to the rate of H+ secretion as measured by pH stat titration.8*, lo Aside from the sodium and H+ pumps, FIGURE 1 depicts an exchange trans-

Mechanisms of K+ transport in isolated turtle urinary bladder. Induction of active K+ secretion in a K+-absorbing epithelium.

Transepithelial K(+) movement was studied in vitro in the short-circuited turtle bladder by increasing luminal K(+) permeability and by inhibiting the basolateral Na/K pump. Luminal addition of amphotericin B caused net K(+) secretion (180+/-52 nmol/h) compared with net K(+) absorption (42+/-6 nmol/h) in control bladders. Serosal ouabain and luminal amiloride abolished K(+) secretion in amphotericin-treated bladders; ouabain restored net absorption (45+/-16 nmol/h). The direction and rate of net K(+) transport are controlled by the relative K(+) permeabilities and the Na/K pump sites at the two cell membranes of the epithelium.

Cellular Defects in Urinary Acidification and Renal Tubular Acidosis

Urinary acidification and renal bicarbonate reabsorption may be altered as a consequence of extrarenal factors or impaired by disease affecting the epithelial cells of the renal tubules and collecting ducts. This chapter is concerned with renal acidifying defects and has as its main focus the disorders of cellular function and membrane transport.