Bicarbonate Flux Research Articles

Effect of dextran-bound acetazolamide on rabbit corneal endothelial ion fluxes: Short Communication Eye

The effects of a carbonic anhydrase inhibitor, acetazolamide, bound to a 72,000 dalton dextran (DBI) on bicarbonate and sodium fluxes across the isolated rabbit corneal endothelium have been examined. When DBI was present on the aqueous-facing endothelial surface, there was a marked inhibition of both the stromal to endothelial unidirectional and net flux of bicarbonate. This suggests that the exit of bicarbonate from the cell into the aqueous-facing solution is influenced by the enzyme carbonic anhydrase. No change was found in sodium fluxes under these same incubation conditions. When DBI was present on the stromal-facing surface of the endothelium, no changes were found in unidirectional or net bicarbonate fluxes; the sodium flux from stroma to endothelium was increased, however, with no change in net flux. This data implies that the link between sodium and bicarbonate movement across the endothelium is not a direct coupling between the transport of the two ions in the form of a symport (Na+:HCO3-) at the apical cell border.

Rheogenic sodium-bicarbonate cotransport in the peritubular cell membrane of rat renal proximal tubule.

The mechanism of bicarbonate transport across the peritubular cell membrane was investigated in rat kidney proximal tubules in situ by measuring cell pH and cell Na+ activity in response to sudden reduction of peritubular Na+ and/or HCO3-. The following observations were made: 1. sudden peritubular reduction of either ion concentration produced the same transient depolarizing potential response; 2. bicarbonate efflux in response to peritubular reduction of bicarbonate was accompanied by sodium efflux; 3. sodium efflux in response to peritubular sodium removal was accompanied by cell acidification indicating bicarbonate efflux; 4. all aforementioned phenomena were inhibited by SITS (10(-3) mol/l) except for a small SITS-independent sodium efflux and depolarization which occurred in response to peritubular sodium removal and was not accompanied by cell pH changes; 5. bicarbonate efflux and accompanying potential changes in response to reduction of peritubular bicarbonate virtually vanished in sodium-free solutions. From these observations we conclude that bicarbonate efflux proceeds as rheogenic sodium-bicarbonate cotransport with a stoichiometry of bicarbonate to sodium greater than 1. The question which of the charged species of the bicarbonate buffer system moves cannot yet be decided. Attempts to determine the stoichiometry from the SITS-inhibitable initial cell depolarization and from the SITS-inhibitable initial fluxes suggest a stoichiometry of 3 HCO3-: 1 Na+. In addition to sodium-dependent bicarbonate flux, evidence was obtained for a sodium-independent transport system of acids or bases which is able to regulate cell pH even in sodium-free solutions.

Facilitated carbon dioxide transport in bovine albumin solutions.

Steady-state CO2 diffusion in the presence of a CO2 gradient was measured in thin layers of bovine albumin solutions containing different amounts of buffer base, added as NaHCO3, and/or of NaCl. In the same solutions, electrical potentials due to the CO2 diffusion across the layers were measured. Addition of carbonic anhydrase induced a chemical reaction equilibrium to exist for the CO2 reaction system, and led to equilibrium values for facilitated CO2 transport due to a bicarbonate flux and to maximum values for the diffusion potential. The diffusion potentials are generated due to the large differences in the ionic mobilities of albumin and other ionic species such as bicarbonate. The diffusion potential markedly reduces the facilitated CO2 flux. The presence of sodium chloride had no significant effect on the CO2 transport rate. The total mass transfer rates of CO2 in albumin solutions were considerably lower than those found by Stroeve and Ziegler (23) in hemoglobin solutions at identical concentrations of buffer base.

Deoxycorticosterone-stimulated bicarbonate secretion in rabbit cortical collecting ducts: effects of luminal chloride removal and in vivo acid loading.

To assess the role of cortical collecting duct bicarbonate secretion in the regulation of net acid excretion, we have sought to identify what factors influence the secretion rate. Net and unidirectional bicarbonate fluxes were measured in isolated perfused cortical collecting ducts from deoxycorticosterone-treated rabbits. The collecting ducts secreted bicarbonate at 11-24 pmol X mm-1 X min-1, confirming the high rate seen in earlier studies. Oral acid loading (50 mM NH4Cl drinking water) completely inhibited the net bicarbonate secretion. The bath-to-lumen flux was markedly reduced with acid loading, but the lumen-to-bath flux changed very little. In tubules from rabbits treated with deoxycorticosterone (but not NH4Cl), luminal chloride replacement with either sulfate or gluconate completely and reversibly inhibited the net bicarbonate secretion. The bath-to-lumen flux was greatly inhibited, but there was little change in the lumen-to-bath flux. We conclude: 1) High rates of bicarbonate secretion can be induced in rabbit cortical collecting ducts by chronic treatment of the animals with deoxycorticosterone. 2) When deoxycorticosterone-treated rabbits were made acidotic by oral administration of NH4Cl, the bicarbonate secretion was prevented, indicating that the systemic acid-base state of the animal may be an important factor regulating bicarbonate secretion. 3) Replacement of chloride in the lumen with sulfate inhibits bicarbonate secretion in the cortical collecting duct, an effect which may explain in part the decrease in urinary pH in response to sulfate infusions in mineralocorticoid-stimulated animals.

Drug effects on the hydraulic conductivity of the isolated rabbit ciliary epithelium.

Three aspects of hydrostatically driven volume flow across the isolated rabbit ciliary epithelium have been examined. (1) Hydraulic conductivity was increased in a dose-dependent manner by amiloride, but not by furosemide, indicating that amiloride may interfere with junctional permeability. The absence of a furosemide effect suggests that regulation of passive fluid movement may be independent of net ionic fluxes of either chloride or bicarbonate. Ethacrynic acid had no effect on hydraulic conductivity at concentrations below 10(-2) M. At 10(-2) M, hydraulic conductivity was decreased, suggesting that fluid pathways may be linked to ionic pathways. (2) Successive in vivo daily treatments with adrenergic agonists result in changes in the intraocular pressure response. Dose-effect curves to in vitro agonists from eyes pre-treated on successive days indicate that isoprenaline caused a lack of response in hydraulic conductivity after 3 d of in vivo treatment, adrenaline caused responses after 2 d which were about half of those found on day 0, while phenylephrine and noradrenaline had no influence on hydraulic conductivity. These data suggest that beta-agonists cause a deactivation or desensitization of some moiety which is coupled with regulation of hydraulic conductivity in a manner similar to that seen for the beta-receptor-coupled adenylate cyclase. (3) While cyclic GMP was without effect, various modifiers of cyclic GMP activity increased hydraulic conductivity. Alteration of cellular cyclic GMP levels appears to exert some regulation over ciliary epithelial permeability.

Effects of benthic macroinvertebrates on the exchange of solutes between sediments and freshwater

Infaunal macrobenthos (tubificid oligochaetes, chironomid larvae, and unionid bivalves) were studied in laboratory microcosms to determine their effects on freshwater sediment diagnesis and the exchange of solutes between sediments and water. Tubificids enhanced the flux of ammonium, bicarbonate, and silica from sediments. After the onset of anoxia, they decreased the flux of iron and phosphate. Chironomids increased the flux of nitrate, bicarbonate, and silica, but did not affect the flux of phosphate. Pore water concentrations were low within the irrigated burrowed zone for chemical species normally high in reduced sediments. Concentration gradients were less steep in the actively irrigated burrow zone, but radial diffusion to and from burrows, increased rates of organic decomposition, and enhanced diatom frustule dissolution rates result in enhanced mass transport from sediments. Data from an experiment with unionid clams demonstrated the presence of radial diffusion gradients. These clams enhanced the chloride and nitrate flux from sediments, decreased the bicarbonate flux, but did not affect the flux of either phosphate or silicate. Although the clams did not actively irrigate their burrows, their effect on sediments was similar to that of chironomids. Comparison of direct and indirect flux estimates showed that both types of estimates could be highly variable. In general, indirect flux estimates were higher than direct flux estimates.

The movement of sodium across short-circuited rabbit corneal endothelium.

Unidirectional sodium and bicarbonate ion fluxes were measured under short-circuit conditions across rabbit corneal endothelium. A net flux of bicarbonate was measured, direction tears to lens, however no net flux of sodium was measured. From these experiments it seems that the inequality between electrically measured short-circuit current and net trans-endothelial bicarbonate ion flux cannot be bridged by any supposed net sodium flux.

Sulfate transport in brush border membrane vesicles prepared from human placental syncytiotrophoblast

Isolated brush-border membrane vesicles prepared from human placenta are known to transport amino acids via a Na +-dependent mechanism akin to that found in gut and kidney vesicle preparations. We studied sulfate transport in placental vesicles and failed to identify any Na +-dependent uptake mechanism. Rather, uptake is a non-electrogenic process that is trans-stimulated by outwardly directed anion flux which is independent of cation. If anion exchange is tightly coupled in vivo , the net transfer of sulfate from mother to the growing fetus may be driven by the continuous flux of bicarbonate in the opposite direction.

Bicarbonate movement across basolateral membrane vesicles from rat jejunum.

The self-orienting Percoll-gradient centrifugation was used to separate basolateral membranes (BLMs) from rat jejunal enterocytes. Bicarbonate uptake into osmotically active BLM vesicles was studied by a rapid filtration technique. The time course of [14C] -labelled bicarbonate uptake was followed for 30 min at 18 degrees C and at pH 8.2. Bicarbonate uptake was fast, not saturable and stimulated by countertransport. This work demonstrates that it is possible to determine bicarbonate flux across BLM vesicles at pH and temperature values close to usual experimental conditions. Together with simple diffusion, bicarbonate would cross the BLM of the enterocyte via a carrier-mediated diffusion process.

The permeability of rabbit and human corneal endothelium.

The fluxes of sodium, chloride and bicarbonate across endothelium plus stroma and then stroma alone were measured in the direction from lens-side to tear-side in rabbit and human corneas in vitro, in order to measure passive permeabilities. The results were used to calculate the permeability of the endothelium. Hodgkin's equation (1951) was then used to calculate the partial electrical conductivity of each ion crossing the endothelium. The summated electrical conductivities of sodium, chloride and bicarbonate were equal to 89 +/- 8% of the measured electrical conductivity, suggesting that the ions diffuse independently across the endothelium in the direction lens-side to tear-side. Stereological analysis of the intercellular spaces supports the idea that the ions permeate through this route and that the physical shape of the spaces determines almost entirely the permeability of the endothelial layer. Trans-endothelial sodium and chloride permeabilities are nearly equal, which may be explained by supposing the intercellular spaces include a cation exchanger of fixed negative charge capacity around 60 m-equiv l.-1 intercellular fluid.

13C]bicarbonate kinetics in humans: intra- vs. interindividual variations.

A comprehensive multicompartmental analysis of HCO-3 kinetics was carried out on five normal, resting, fasted adults on three separate occasions at 1-wk to 1-mo intervals to obtain a set of bicarbonate kinetic parameters and estimates of their inter- and intraindividual variations for use in the design and analysis of future nutrient oxidation studies. Following a rapid bolus of NaH13CO3 (10 mumol . kg-1 iv), the decay of 13C enrichment of breath CO2 could be described by a three-exponential decay process and a linearly time-dependent term that accounted for changes in the 13C enrichment of metabolic fuels. The data were fitted subsequently to a mammillary multicompartmental model that consisted of a central pool and two peripheral pools of 3,310, 3,490, and 8,070 mumol . kg-1 HCO-3-CO2. Labeled CO2 was eliminated from the central pool by respiratory and nonrespiratory routes at rates of 101 and 97 mumol . kg-1 . min-1, respectively. The within-subject and among-subject variances were similar for the amount of freely exchangeable bicarbonate (14,870 mumol . kg-1), CO2 output (101 mumol . kg-1 . min-1), bicarbonate flux (198 mumol . kg-1 . min-1), and the fraction of administered bicarbonate recovered in breath (0.51). Comparison of variances associated with the assignment of a population mean value to a new subject and the variance associated with the use of a value obtained in the same individual on another day indicated that there was no advantage to making a determination on an alternate day over using a population value. Efforts should be made to compile population values for bicarbonate kinetics in different age groups and metabolic, nutritional, and pathological states for use in interpretation of nutrient oxidation data.

Characteristics of bicarbonate, sodium, and chloride fluxes in the rabbit corneal endothelium

Unidirectional fluxes of bicarbonate, sodium and chloride were measured across the isolated rabbit corneal endothelium. Bicarbonate and sodium fluxes were measured between 13 and 37°C and were found to be temperature-sensitive. Ouabain (10 −6 m), which inhibits endothelial fluid transport, reduced the net bicarbonate flux by 32% but had no significant effect on sodium fluxes. Amiloride (10 −4 m) did not alter sodium transport, but slightly increased the unidirectional bicarbonate fluxes. Sodium fluxes were unchanged in chloride-free and potassium-free Ringer solutions, and net sodium transport was unaffected in the presence of carbonic anhydrase, acetazolamide, or low (5 m m) bicarbonate Ringer, but was reduced by 45% in bicarbonate-free Ringer. A net chloride flux was found in the same direction (stroma to aqueous) as bicarbonate and sodium. This chloride flux (2·18 μEq/cm 2/hr) was of the same magnitude as bicarbonate but was sodium-independent. These findings suggest that: (1) like bicarbonate and fluid transport, sodium transport is bicarbonate-dependent, temperature-sensitive, and chloride-independent; (2) sodium transport is not stoichiometrically coupled to bicarbonate; and (3) neither sodium nor bicarbonate transport are coupled to that of chloride.

Bicarbonate uptake by basolateral membrane vesicles from rat jejunum.

Basolateral membranes from rat jejunal enterocytes have been obtained by self-orienting Percoll-gradient centrifugation. Bicarbonate and L-glucose uptake into osmotically active basolateral membrane vesicles has been studied by a rapid filtration technique. In closed vessels and at pH 8.2 the uptake kinetics of both [14C]bicarbonate and L[3H]glucose have been followed for 30 min at 18 degrees C. Bicarbonate uptake seems to be fast and in efflux experiments SITS and DIDS effect is negligible. This work demonstrates that it is possible to determine bicarbonate flux across basolateral membrane vesicles at pH and temperature values close to usual experimental conditions.

Hydrogen ion balance in dialysis therapy.

A model to describe hydrogen ion balance (H+B) in acetate and bicarbonate dialysis therapy was developed based on measurement of metabolic addition of hydrogen ion (H+) to the body between and during dialyses and measurement of net buffer repletion during dialysis. Metabolic H+ generation was shown to be equal to 0.77 times the protein catabolic rate plus the total net removal of lactate and beta-hydroxybutyrate ions during dialysis. Buffer repletion was calculated from total net flux of acetate and bicarbonate during dialysis. The model was used for eight paired studies of H+B on one week each of acetate and bicarbonate dialysis and showed that cumulative H+B with acetate was -7 +/- 28 (M +/- SEM) mmol/week compared to -175 +/- 45 mmol/week with bicarbonate (P less than 0.001). It is concluded that there is an initial, strongly negative H+B when patients on acetate dialysis are converted to bicarbonate. The possible physiologic significance of this is discussed.

Mathematical model of the alkalization of suspensions of the alga Hydrodictyon reticulatum in light

The assumption that HCO 3 − ions are taken up in exchange for OH − ions in a 1:1 ratio can be used to explain the exponential time course of pH change in suspensions of the alga Hydrodictyon reticulatum during the light phase. Using two starting equations, one expressing the dependence of the suspension pH on time, the other the dependence of pH-stat flow on pH, and taking into consideration basic conservation laws, the following were calculated: concentrations of bicarbonate and carbonate anions, the flux of carbon dioxide into the system from the air and bicarbonate flux into the cells.

495—Mathematical model of the alkalization of suspensions of the alga Hydrodictyon reticulatum in light

The assumption that HCO 3 − ions are taken up in exchange for OH − ions in a 1:1 ratio can be used to explain the exponential time course of pH change in suspensions of the alga Hydrodictyon reticulatum during the light phase. Using two starting equations, one expressing the dependence of the suspension pH on time, the other the dependence of pH-stat flow on pH, and taking into consideration basic conservation laws, the following parameters were calculated: concentrations of bicarbonate and carbonate anions, the flux of carbon dioxide into the system from the air and bicarbonate flux into the cells.

A reciprocating, single-needle hemodialyzer with bidirectional flow of sorbent suspension.

Previous theoretical analysis has indicated that adequate mass transfer is possible in a dialyzer with reciprocating membrane motion provided that the dialysate concentration of uremic substances is kept low. Earlier models have utilized a collection of sorbents (charcoal, urease, and a cation exchanger) constrained next to the dialyzer membranes. We have designed a new dialyzer with a sorbent suspension having free access from a reservoir to the spaces between membrane packages. At a treatment rate of 150 ml/min/m2, the in vitro creatinine clearance is 75 ml/min/m2, which agrees within experimental accuracy with the theoretical prediction. The creatinine clearance, flow resistance, and compliance of the dialyzer are constant during four to six hours of testing. In vivo tests have been performed during urea and creatinine infusion in a normal dog and in a dog with 3/4 nephrectomy. The in vivo creatinine clearance agrees within 10% with the in vitro clearance. Sodium, potassium, calcium, and bicarbonate fluxes are acceptable for patients in renal failure. The new design allows a higher capacity for urea and creatinine, since larger amounts of sorbent may be used.

Rabbit corneal endothelial permeability in the presence and absence of adenosine and glutathione.

Rabbit corneal endothelial fluxes of sodium, bicarbonate, inulin, and dextran have been measured in ambient solutions containing either 0.5 mM adenosine alone (A), 0.3 mM glutathione alone (G), both adenosine and glutathione, or neither adenosine nor glutathione. Addition of A alone to a solution lacking both additives caused a decrease in both Jstrendo and Jendostr sodium fluxes coupled with an increase in Jstrendo net. Addition of G alone caused no effect, whereas addition of A and G together caused a decrease in both Jstrendo and Jendostr sodium fluxes and a large increase in Jstrendo net. Addition of A or G caused an increase in Jstrendo bicarbonate flux, but no change in Jendostr flux. Addition of A and G caused an increase in both Jstrendo and Jendostr bicarbonate flux and a decrease in Jstrendo net. None of the solution variations caused an alteration in either inulin or dextran permeability. Since both A and G influence Jstrendo net bicarbonate and sodium transport, this may provide an explanation of the beneficial effects of these compounds on the endothelium to both enhance and prolong corneal thickness and hydration regulation.

Carbonic anhydrase independent bicarbonate reabsorption.

The present study was designed to define the prerequisites of carbonic anhydrase independent bicarbonate reabsorption. In free flow experiments during systemic application of carbonic anhydrase inhibitor benzolamide (50 mg/kg B. W.) bicarbonate recovery in % of filtered load was found to be 74 +/- 8% in late proximal convoluted tubules, 39 +/- 6% in distal convoluted tubules and 32 +/- 4% in urine, indicating that most of carbonic anhydrase independent bicarbonate reabsorption occurs in tubule segments prior to distal convoluted tubules. In vivo continuous microperfusion experiments in proximal convoluted tubules demonstrated that luminal benzolamide (0.5 mmol/l) virtually abolishes net bicarbonate fluxes, when bicarbonate concentration in the luminal perfusate (25 mmol/l) is close to peritubular plasma concentration (24.4 mmol/l). In contrast, a significant downhill reabsorptive flux occurs, when perfusate bicarbonate concentration is 75 mmol/l and a significant downhill secretory flux is observed, when the perfusate is initially free of bicarbonate. The corresponding apparent permeabilities are 1.0 +/- 0.1 X 10(-6) cm2/s for influx and 1.6 +/- 0.4 X 10(-6) cm2/s for efflux of bicarbonate. Clearance studies reveal that carbonic anhydrase dependent and independent bicarbonate reabsorption are not saturable but depend on the rate of volume reabsorption in the kidney. In conclusion, passive movements of bicarbonate do occur in proximal convoluted tubules and most likely contribute to carbonic anhydrase independent bicarbonate reabsorption.

Photodynamic alteration of cornea endothelium: Relation to bicarbonate fluxes and oxygen concentration

Corneas were mounted in flux chambers and endothelial bicarbonate fluxes were determined following sensitization of endothelial cells with 5 · 10 −6 M rose bengal and exposure to light. Corneas exposed to light demonstrated an increased passive bicarbonate flux compared to corneas not photosensitized. Active bicarbonate flux was reduced after 5 min of light exposure, but not after 1 min of light exposure. The increase in passive bicarbonate flux was prevented by the addition of 200 μg/ml catalase to the bathing solution; however, catalase had no effect on the photodynamic alteration of active flux. Neither 10 mM ascorbic acid nor 1.012 g/l glutathione prevented the photodynamically induced increase in passive flux. Perfusion of corneas with 5 · 10 −6 M rose bengal dissolved in a sucrose-substituted Krebs-Ringer bicarbonate solution with a p o 2 of 124 ± 4.0 mmHg and exposed to light swelled at rates more rapid than corneas treated in a similar fashion but perfused with a solution with a P o 2 of 20 ± 4.6 mmHg . This study demonstrates that photodynamically induced corneal endothelial cell alteration results in increased passive bicarbonate flux, a time-dependent decrease in active bicarbonate flux, is oxygen dependent, and is at least in part secondary to H 2O 2 produced by the dismutation reaction of the superoxide free radical.