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The apical cell membranes of the H+ secreting, alpha-intercalated cells of turtle urinary bladder (TB) are characterized by studs (cytoplasmic domains of V-adenosinetriphosphatase) on thin-section transmission electron microscopy and by intramembrane particles (spherical units, SPUs) occurring as rod-shaped particles on freeze-fracture electron microscopy. To examine the relationship between studs and SPUs, morphometric studies were carried out on bladders maintained in 5% CO2 and in the absence of exogenous CO2. The stud density per square micrometer of apical membrane was 3,909 +/- 352 (+/-SE) in four TBs (29 alpha-cells) at 5% CO2 and 3,667 +/- 448 (+/-SE) in the paired halves of the same bladders without CO2 (25 alpha-cells). Corresponding densities of SPUs counted on apical membranes of the same bladders (n = 4) were 3,941 +/- 545 in 5% CO2 and 3,599 +/- 511 without CO2. The similarity of the densities of studs and SPUs under both conditions indicates that each SPU within the membrane is matched by one stud projecting into the cytoplasm. The one-for-one relationship between studs and SPUs was preserved over a wide range of transport rates. Addition of CO2 caused only inconsistent increments in the densities of studs and SPUs despite substantial increases in H+ transport rate. Slight variations in spacing of studs were consistent with patterns of distribution of SPUs on fracture surfaces.

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Since the time of Smith, studies of urinary acidification have shifted their focus to ever smaller scales and have revealed iterative patterns or organization. For this review we focus on the organization of intra- and submembrane particles at the scale of the apical cell membrane of the H+ secreting, alpha intercalated cells. Particles were examined quantitatively by thin section and freeze-fracture (FF) electron microscopy. Ongoing studies in turtle bladder indicate that the density of submembrane particles (studs) per micron 2 is approximately the same as that of spherical units (SPUs) forming linear (rod-shaped) arrays on FF. This one-to-one relationship is observed in the presence or absence of CO2 and suggests that CO2-induced changes in H+ secretion do not involve dissociation of the intramembrane (channel) and cytoplasmic (catalytic) parts of the H-ATPase. Structure-function studies based on density estimates of the particles, morphometry of the H+ secreting cell population, and measurement of H+ transport rate prior to fixation permit functional correlation across scales of study.

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To explore the possible contribution of an H-K-adenosine-triphosphatase (H-K-ATPase) to H+ secretion (JH) in the isolated turtle bladder, we measured electrogenic JH (JeH) as short-circuit current and total JH (JTH) by pH stat titration in the presence of ouabain at different ambient K+ concentration ([K+]) and during luminal addition of a known gastric H-K-ATPase inhibitor, Schering (Sch)-28080. JH was not reduced by decreasing ambient [K+] to undetectable or very low levels (< 0.05 mM by atomic absorption) and luminal BaCl2 addition to further reduce local [K+] at the apical membrane. These K(+)-removal studies indicate that H+ transport is not coupled to countertransport of K+. JTH did not exceed JeH at any point: in K(+)-free solutions JTH was 0.73 +/- 0.05, and JeH was 0.95 +/- 0.08 mumol/h; in standard (3.5 mM) K+ solutions JTH was 0.72 +/- 0.05 and JeH 0.98 +/- 0.06 mumol/h; in high (118 mM) K+ solutions JTH was 0.65 +/- 0.07 and JeH 0.94 +/- 0.08 mumol/h. Sch-28080 caused a rapid inhibition of JH, with similar half-maximal inhibitory concentrations (IC50) in K(+)-free, standard [K+], and high [K+] solutions. Bafilomycin inhibited JeH and JTH with an IC50 of approximately 100 nM. The observed non-potassium-competitive inhibition of JH by Sch-28080 and the bafilomycin sensitivity distinguish the H-ATPase of the turtle bladder from the gastric H-K-ATPase. The rapidity of the inhibition by Sch-28080 suggests that it acts at an accessible luminal site of the ATPase.

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The apical anion exchanger of the beta-carbonic anhydrase (CA) cells differs from the basolateral exchanger of the alpha-cells by reduced sensitivity to disulfonic stilbenes and lack of immunoreactivity with antibodies to erythrocyte band 3 protein. To characterize the exchanger, we examined the effects on electroneutral bicarbonate secretion (JHCO3n) of Cl- replacement by gluconate, Br-, SO4(2-), and NO3- and of inhibition by 1) acetazolamide (ACZ) with and without pretreatment with sodium azide (NaN3), 2) furosemide, and 3) alpha-cyano-4-hydroxycinnamate (CHC). The Cl-dependent JHCO3n was 0.90 +/- 0.09 mumol/h, similar to the ACZ-inhibitable rate of 0.83 +/- 0.08 mumol/h with an apparent Km for Cl near 3.4 mM. Maximal JHCO3n was comparable at luminal pH 6.8 and 4.5. JHCO3n was reduced to approximately 21% in Br-, 13% in SO4(2-), and 7% in NO3- solutions compared with the rates in chloride solutions. ACZ inhibition was not abolished by pretreatment with NaN3. JHCO3n was only slightly inhibited (14%) by furosemide and not inhibited by CHC. In conclusion, the apical exchanger is selective for chloride and relatively resistant to inhibitors. Its dependence on luminal chloride is such that its transport rate is closely regulated by mucosal chloride at concentrations below 20 mM.

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The uptake of the fluorescent anion, N-(7-nitrobenz-2-oxa-1,3 diazol-4-yl)aminoethanosulfonic acid-taurine (NBD-T) was examined in sheets of turtle bladder epithelial cells after either serosal or mucosal exposure to the fluorescent anion. Serosal uptake of NBD-T into a population of epithelial cells was demonstrated in thin epithelial sheets scraped from bladder sacs exposed to low (0.5 mM) concentrations of NBD-T sufficient to cause fluorescence in turtle erythrocytes. In nine sheets NBD-T fluorescence was observed in 14.4 +/- 1.3% of epithelial cells; by phase-contrast microscopy of the same fields, these cells were nongranular or carbonic anhydrase (CA) cells. An additional 8.1 +/- 1.0% of cells was nongranular, but failed to exhibit fluorescence after serosal NBD-T exposure, consistent with a beta-CA cell population. Apical uptake of NBD-T into epithelial cells was demonstrable only at high (5 mM) mucosal concentrations in both thin sheets and superfused bladder segments. In thin sheets, NBD-T fluorescence was observed in 15.8 +/- 0.7% of cells and appeared to have a punctate subapical distribution; phase-contrast of the same fields (n = 46) revealed a nongranular cell population of 21.9 +/- 0.9%. Nongranular cells corresponded closely to CA cells as identified with carboxyfluorescein fluorescence. Hence, apical NBD-T was taken up primarily by alpha-CA cells. In conclusion, NBD-T is transported by alpha-CA cells either across the basolateral membrane with high affinity comparable to that of turtle red cell band 3 protein or across the apical membrane with low affinity via a less-specific mechanism such as endocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)

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1. The urinary acification system present in the bladder of the toad Bufo marinus ictericus was investigated by means of an improved technique of mounting the membrane that minimizes the edge damage when the bladder is placed as a sheet between lucite half chambers. 2. In ouabain-treated bladders in the absence of exogenous CO2, acidification rates were similar to those observed for turtle bladder. 3. The presence of 1% CO2 at the serosal gas phase increased proton secretion (JH). Stimulation of JH was also observed when the cell pH was decreased by back diffusion of salicylic acid added to the mucosal (M) compartment. 4. The estimate of the passive proton permeability of this epithelium as a whole yielded values around 1 x 10(-4) cm/s. The maximum pH gradient that could be established across the membrane in the short circuit condition (about 3 pH units) was taken as the apparent proton-motive force (PMF') of the system and these values were similar to those observed in the turtle bladder. 5. Luminal membrane depolarization caused by substitution of NaCl by KCl in Ringer solution led to an increase in JH at M pH = 7.3 without altering the PMF', which suggests that the electrical potential difference across the luminal membrane in ouabain-treated bladders is negligible when M pH is sufficiently acid to abolish H+ secretion.

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The urinary acidification system present in the bladder of the toad Bufo marinus ictericus was investigated by means of an improved technique of mounting the membrane that minimizes the edge damage when the bladder is placed as a sheet between lucite half chambers. In ouabain-treated bladders in the absence of exogenous CO2, acification rates were similar to those observed for turtle bladder. The presence of 1%CO2 at the serosal gas phase increased proton secretion (JH). Stimulation of JH was also observed when the cell pH was decreased by back diffusion of salicylic acid added to the mucosal(M) compartment. The estimate of the passive proton permeability of this epithelium as a whole yielded values around 1 x 10**-4cm/s. The maximum pH gradient that could be established across the membrane in the short circuit condition (about 3 pH units) was taken as the apparent proton-motive force (PMF') of the system and these values were similar to those observed in the turtle bladder. Luminal membrane depolarization caused by substitution of NaCl by KCl in Ringer solution led to an increase in JH at MpH = 7.3 without altering the PMF', which suggests that the electrical potential difference across the luminal membrane in ouabain-treated bladders is negligible when M pH is sufficiently acid to abolish H+ secretion

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Structure-function studies of the turtle bladder indicate that electrogenic proton secretion into the urinary compartment is accomplished by alpha-type intercalated cells which are rich in carbonic anhydrase. In the absence of electrochemical potential gradients (delta mu H = 0), the rate of H+ secretion (JH) is a function of the number of H+ pumps in position at the apical cell membrane, as judged from morphometric and freeze-fracture studies of apical membrane area characterized by a cytoplasmic coating with studs and by rod-shaped intramembrane particles (RSP). At a given pump population, JH is a sigmoid function of delta mu H, with delta pH and delta psi having equivalent effects on JH. The JH versus delta mu H relation reflects the intrinsic properties of the H+ pump and suggests a H+ pump model consisting of two components, a channel through the apical membrane across which delta mu H falls, and a catalytic unit located within the cytoplasm (outside of delta mu H). Each intramembrane RSP is associated with several cytoplasmic studs, but the precise relations between the two remain to be clarified.

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The turtle bladder contains transport systems for active sodium absorption, electrogenic proton secretion, and bicarbonate secretion (coupled to chloride absorption) that are functionally separate and occur in specialized epithelial cells. Maneuvers that alter the intracellular acid-base state, such as changes in PCO2, cause marked changes in the apical membrane area of alpha-type carbonic anhydrase (CA) cells by addition or retrieval of membrane vesicles but have no effect on the granular cells that transport sodium. The apical cell membrane of alpha-CA cells contains characteristic rod-shaped intramembrane particles (RSP) by freeze fracture and is coated on its cytoplasmic side with studs. A subpopulation of CA cells (beta-type), which is characterized by apical microvilli, fails to exhibit an apical response to CO2 stimulation and does not reveal RSPs or studs at its apical membranes; instead, these elements can be demonstrated at the basolateral membrane. The reversal in the polarity of these elements as well as physiological evidence suggest that beta-type cells are responsible for bicarbonate secretion. Structure-function studies of CO2 stimulation of H+ secretion by alpha-CA cells indicate that the secretion rate (JH) correlates with apical membrane area and numbers of RSPs. The view that RSPs represent arrays of transmembrane channels and that studs represent catalytic units of H+ pumps is supported by quantitative considerations but remains to be proven. Urinary acidification is regulated not only by changes in the number of H+ pumps but also by the intrinsic properties of the H+ pump itself. For a given pump population, JH is closely controlled by the delta microH across the active transport pathway.

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To correlate the prevalence of rod-shaped intramembrane particles (RSP) in the apical membranes of carbonic anhydrase-rich (CA) cells and the H+ transport rate in turtle urinary bladder, we carried out morphometric studies by means of scanning and freeze-fracture electron microscopy of the alpha and beta subpopulations of CA cells. Correlations were made between the apical membrane areas of alpha cells and H+ transport rate at 0 and 5% ambient CO2. Exposure to CO2 more than doubled the planar area of the luminal surface of alpha cells and increased the degree of folding (amplification) of the apical cell membrane from 2.8 +/- 0.3 to 3.8 +/- 0.3. The actual apical membrane area of alpha cells increased from 176 mm2 to 693 mm2 per 8 cm2 epithelial area. The RSP density also appeared to be increased by about 40%. The total CO2-induced increase in RSPs in position at the luminal surface was 5 fold while the increase in H+ transport was 9-fold. We conclude that stimulation of H+ transport by CO2 involves recruitment of RSP to the apical cell membrane of alpha-type CA cells and that RSPs are associated with active H+ transport. They may represent linear arrays of transmembrane components of H+ pumps.

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To define the transport pathway for HCO-3 secretion (JHCO3) across the apical and basolateral membranes of turtle bladder, we examined the effects of cAMP, isobutylmethylxanthine (IBMX), the Cl- channel blocker 9-anthroic acid (9-AA), and the disulfonic stilbene DIDS (4,4'-diisothiocyanostilbene-2,2'-sulfonic acid) on the electroneutral and electrogenic components of JHCO3. Total JHCO3 was measured by pH stat titration of the mucosal compartment after Na+ absorption and H+ secretion were abolished by ouabain and a delta pH, respectively. Addition of cAMP or IBMX increased total JHCO3 and induced a short-circuit current (ISC), accounting for a large part of JHCO3; net Cl- absorption was reduced. Mucosal 9-AA inhibited the IBMX-induced electrogenic component of JHCO3, whereas mucosal DIDS inhibited the electroneutral component and acetazolamide reduced both. We suggest that HCO-3 is generated within the cell by a Na-independent primary active acid-base transport at the basolateral membrane (H+ extrusion into the serosal compartment). Cellular HCO-3 accumulation drives JHCO3 via a Cl-HCO3 exchanger at the luminal membrane. IBMX and cAMP activate a 9-AA-sensitive anion conductance parallel to the exchanger. The apparent reversal of the transport elements between the two cell membranes (compared with H+-secreting cells) led to an ultrastructural examination of the carbonic anhydrase-rich cells.

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The carbonic anhydrase-rich (CA) cell population of the turtle urinary bladder, which is responsible for the secretion of H+ and probably of HCO-3, was studied by freeze-fracture and thin-section electron microscopy. The apical membrane of the major CA cell type (alpha type) was characterized by microplicae and by a coat of studs on its cytoplasmic side; on freeze-fracture, it contained a dense population of rod-shaped intra-membrane particles. When fixed at low CO2 tension, the apical membrane area of the alpha cell was reduced; its surface displayed microplicae as well as microvilli, and the apical cytoplasm contained many vesicles with rod-shaped particles and studs. The apical membrane of the other (beta type) CA cell was characterized by numerous individual microvilli without microplicae and by a relative absence of rod-shaped particles and studs. Instead, the beta cell contained studs and rod-shaped particles in its basolateral membrane. The ultrastructure and frequency of the beta CA cell were not affected by changes in CO2 tension. We suggest that the alpha cell is responsible for H+ secretion. The reversal of the polarity of the membrane elements in the beta cell and failure to respond to CO2 with amplification of its apical membrane are consistent with a role in HCO-3 secretion.

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A number of tight urinary epithelia, as exemplified by the turtle bladder, acidify the luminal solution by active transport of H+ across the luminal cell membrane. The rate of active H+ transport (JH) decreases as the electrochemical potential difference for H+ [delta mu H = mu H(lumen) - mu H(serosa)] across the epithelium is increased. The luminal cell membrane has a low permeability for H+ equivalents and a high electrical resistance compared with the basolateral cell membrane. Changes in JH thus reflect changes in active H+ transport across the luminal membrane. To examine the control of JH by delta mu H in the turtle bladder, transepithelial electrical potential differences (delta psi) were imposed at constant acid-base conditions or the luminal pH was varied at delta psi = 0 and constant serosal PCO2 and pH. When the luminal compartment was acidified from pH 7 to 4 or was made electrically positive, JH decreased as a linear function of delta mu H as previously described. When the luminal compartment was made alkaline from pH 7 to 9 or was made electrically negative, JH reached a maximal value, which was the same whether the delta mu H was imposed as a delta pH or a delta psi. The nonlinear JH vs. delta mu H relation does not result from changes in the number of pumps in the luminal membrane or from changes in the intracellular pH, but is a characteristic of the H+ pumps themselves. We propose a general scheme, which, because of its structural features, can account for the nonlinearity of the JH vs. delta mu H relations and, more specifically, for the kinetic equivalence of the effects of the chemical and electrical components of delta mu H. According to this model, the pump complex consists of two components: a catalytic unit at the cytoplasmic side of the luminal membrane, which mediates the ATP-driven H+ translocation, and a transmembrane channel, which mediates the transfer of H+ from the catalytic unit to the luminal solution. These two components may be linked through a buffer compartment for H+ (an antechamber).

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To characterize the efflux of HCO-3 across the basolateral membrane of the H+-secreting cells of the turtle bladder, we examined the effect of substitution of gluconate or methyl sulfate for Cl- on the rate of acidification (JH). JH was measured as the short-circuit current in bladders in which Na+ transport was abolished with 10(-4) M ouabain. In hemibladders bathed in normal Ringer solution (Cl- = 122 mM) JH was 44.9 microA. Substitution of the Cl- resulted in a marked reduction in JH (12.5 microA with gluconate and 7.5 microA with methyl sulfate). Addition of Cl- to the mucosal surface had no effect on JH. In contrast, serosal addition of Cl- restored JH to control. The apparent Km for Cl- in gluconate Ringer was 0.13 mM. Serosal furosemide (1 mM) inhibited JH by 55% in Cl- Ringer. We conclude that HCO-3 exit across the basolateral membrane of the H+-secreting cell occurs via a Cl-HCO3 exchanger that has a high affinity for chloride.

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This article has examined the process of urinary acidification from the perspective of events occurring at the cellular and single nephron level. Accordingly, the reabsorption of filtered HCO3- and the titration of urine buffers can be ascribed to the fundamental process of H+ secretion. The precise mechanism of H+ secretion by the tubule cells, the rate by which this occurs, and the factors regulating transport differ between nephron segments. Despite these differences, the cellular process of urinary acidification can be viewed as the extrusion of H+ against an electrochemical gradient across the luminal cell membrane and the movement of an HCO3- equivalent across the basolateral cell membrane. In the proximal tubule (convoluted and straight portions) approximately 90 per cent of the filtered load of HCO3- is reabsorbed. This occurs without the development of large lumen-to-blood pH gradients. The secretion of H+ across the luminal membrane occurs primarily via an electrically neutral Na-H exchange mechanism. Since it is the lumen-to-cell Na+ gradient which provides the energy, the secretion of H+ is a "secondary active" process dependent on the function of the Na-K-ATPase located in the basolateral cell membrane. During the elaboration of an acid urine, the distal nephron (distal convoluted tubule and collecting duct) reabsorbs that portion of the filtered HCO3- escaping proximal reabsorption, titrates luminal buffers, and lowers urine pH. The secretion of H+ occurs by a "primary active" mechanism, which involves the extrusion of H+ across the luminal cell membrane by an electrogenic H+ pump driven by the hydrolysis of ATP. The rate at which H+ is secreted depends on the electrochemical gradient for H+ across the luminal membrane. Thus, changes in both lumen pH and potential will effect H+ secretion, with low lumen pH inhibiting transport and large lumen-negative potentials stimulating transport. In some animals, depending on their homeostatic needs, secretion of HCO3- by the distal nephron can also occur. This process is localized to the distal convoluted tubule and the cortical collecting duct and appears to represent a transport system distinct from that responsible for H+ secretion.

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The changes in cell structure produced during stimulation of proton secretion by CO2 in turtle bladder were examined using ultrastructural morphometric methods. One hour after CO2 addition, the area of the luminal membrane of the carbonic anhydrase-containing (CA) cell population was increased 2.5-fold and the volume percent of electron-lucent cytoplasmic vesicles in these CA cells was decreased by 61%. No changes were observed in the epithelial granular cells. These results suggest that during CO2 stimulation the vesicles fuse with the luminal membrane. CO2 stimulation of proton secretion is inhibited by the cytoskeleton-disrupting drugs colchicine and cytochalasin B and by 99% deuterium oxide as the Ringer solvent. Deuterium oxide also inhibits the decrease in cytoplasmic vesicles. Thus stimulation of proton secretion by turtle bladder CA cells depends to a large extent on vesicle fusion and the resultant increase in luminal surface area.

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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.

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