Evidence for HCO3− conductance pathways in nutrient membrane of bullfrog antrum

The effect of changing the nutrient-side HCO₃⁻ concentration on potential difference (PD) and resistance in bullfrog antrum bathing in Cl⁻ media was determined. Changes in HCO₃⁻ concentration were from 25 mM to several lower concentrations and back to 25mM. A plot of 6ΔPD6 versus log [HCO₃⁻] gave a linear relation for changes of HCO₃ concentration from 25 down to 3.1 mM and back to 25 mM but deviated to some extent for changes to 1.6 mM. In these experiments, changes from higher to lower HCO₃⁻ concentrations gave a less rapid initial PD response than those in the reverse direction. This result eliminated H⁺ conductance pathways as being predominant. Experiments were done in which in the first part changes were made in nutrient solution from 5% CO₂ and 25 mM HCO₃⁻ to 0.6% CO₂ and 3 mM HCO₃⁻ and in the second part the same changes with a simultaneous change of secretory solution from 5% to 10% CO₂. The magnitude of PD decrease was greater by 4.5 mV in the second part. This result indicated that HCO₃⁻ conductance pathways rather than OH⁻ conductance pathways predominated. There was no evidence of HCO₃⁻, OH⁻ and H⁺ conductance pathways in secretory membrane.     

Cellular mechanism of HCO 3 − and Cl− transport in insect salt gland

Summary Active HCO ₃ ^t- secretion in the anterior rectal salt gland of the mosquito larva,Aedes dorsalis, is mediated by a 11 Cl^–/HCO ₃ ^– exchanger. The cellular mechanisms of HCO ₃ ^– and Cl^– transport are examined using ion- and voltage-sensitive microelectrodes in conjunction with a microperfused preparation which allowed rapid saline changes. Addition of DIDS or acetazolamide to, or removal of CO₂ and HCO ₃ ^– from, the serosal bath caused large (20 to 50 mV) hyperpolarizations of apical membrane potential (V _a) and had little effect on basolateral potential (V _bl). Changes in luminal Cl^– concentration alteredV _a in a repid, linear manner with a slope of 42.2 mV/decaloga _Cl ^l –. Intracellular Cl^– activity was 23.5mm and was approximately 10mm lower than that predicted for a passive distribution across the apical membrane. Changes in serosal Cl^– concentration had no effect onV _bl, indicating an electrically silent basolateral Cl^– exit step. Intracellular pH in anterior rectal cells was 7.67 and the calculated was 14.4mm. These results show that under control conditions HCO₃ enters the anterior rectal cell by an active mechanism against an electrochemical gradient of 77.1 mV and exits the cell at the apical membrane down a favorable electrochemical gradient of 27.6 mV. A tentative cellular model is proposed in which Cl enters the apical membrane of the anterior rectal cells by passive, electrodiffusive movement through a Cl^–-selective channel, and HCO ₃ ^– exits the cell by an active or passive electrogenic transport mechanism. The electrically silent nature of basolateral Cl^– exit and HCO₃ entry, and the effects of serosal addition of the Cl^–/HCO₃ exchange inhibitor, DIDS, on and transepithelial potential (V _ic) suggest strongly that the basolateral membrane is the site of a direct coupling between Cl^– and HCO ₃ ^– movements.     

HCO 3 − /Cl− exchange across the human erythrocyte membrane: Effects of pH and temperature

Summary Changes in extracellular pH (pH_o) in red cell suspensions were monitored in a stopped-flow rapid reaction apparatus under conditions wheredpH_o/dt was determined by the rate of HCO ₃ ^– /Cl^– exchange across the membrane. Experiments were performed at 5°C<T<40°C using either untreated cells or cells exposed to 0.11mm SITS (4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid). Although SITS exposure reduced the rate of exchange by 90%, both untreated and SITS-treated cells are similarly affected by changes in pH₀ and temperature. The rate of HCO ₃ ^– /Cl^– exchange exhibits a minimum at about pH_o 5 and a maximum at about pH₀ 7.4 at all temperatures. A transition temperature of 17°C was observed in the Arrhenius relationship for all pH₀. The activation energies (E _a) in kcal/mol are 19.6 below and 11.7 above 17°C for 50<8. These findings, similar to those reported for Cl^– self-exchange, suggest that: (i) a change in the rate-limiting step for HCO ₃ ^– /Cl^– exchange occurs at 17°C, possibly due to an altered interaction between the transport pathway and membrane lipids; (ii) the carrier system can be titrated by either H⁺ or SITS from the outside of the membrane, but the untitrated sites continue to transport normally; (iii) the pH₀ dependence of the rate of exchange is consistent with the titratable carrier having its most alkaline pK in the range expected for amino groups; and (iv) below pH₀ 5, the nature of the exchange is markedly altered.     

Potential difference responses to secretory K+, Na+ and HCO3− changes in secreting and resting states of frog stomach in Cl−-free media

The effects of changes in secretory concentrations of K⁺, Na⁺ and HCO₃⁻ on transmucosal potential difference (PD) and resistance in Cl⁻-free (SO₄²⁻) solutions were compared for secreting fundus and resting fundus of Rana pipiens. In the resting fundus experiments, histamine was not present in the nutrient solution and cimetidine was primarily used to obtain acid inhibition. Increase of K⁺ from 4 to 80 mM, decrease of Na⁺ from 156 to 15.6 mM and decrease of HCO₃⁻ from 25 to 5 mM gave, 10 min after the change, in the secreting fundus Δ PD values of 39.7, −11.9 and 3.2 mV, respectively. In the resting fundus, 1.5 to 2 h after the addition of cimetidine, the same changes in secretory ion concentration gave Δ PD values of 12.2, −5.6 and 1.5 mV, respectively. Replacement of cimetidine with SCN and without histamine yielded a Δ PD somewhat lower than that in cimetidine, namely 9 mV for a K⁺ change from 4 to 80 mM. Subsequent addition of histamine with SCN present gave a Δ PD of about 21 mV. The change in PD was attributed to histamine increasing the secretory membrane area, leading to an increase in K⁺ conductance. Another possibility is that histamine increases the K⁺ conductance per se.     

Secretion of HCO 3 − /OH− in cortical distal tubule of the rat

Secretion of bicarbonate has been described for distal nephron epithelium and attributed to apical Cl^–/HCO ₃ ^– exchange in beta-intercalated cells. We investigated the presence of this mechanism in cortical distal tubules by perfusing these segments with acid (pH 6) 10 mm phosphate Ringer. The kinetics of luminal alkalinization was studied in stationary microperfusion experiments by double-barreled pH (ion-exchange resin)/1 m KCl reference microelectrodes. Luminal alkalinization may be due to influx (into the lumen) of HCO ₃ ^– or OH^–, or efflux of H⁺. The magnitude of the Cl^–/ HCO ₃ ^– exchange component was measured by perfusing the lumen with solutions with or without chloride, which was substituted by gluconate. This component was not different from zero in control and alkalotic (chronic plus acute) Wistar rats. Homozygous Brattleboro rats (BRB), genetically devoid of antidiuretic hormone, were used since this hormone has been shown to stimulate H⁺ secretion, which could mask bicarbonate secretion. In these rats, no evidence for Cl^–/HCO ₃ ^– exchange was found in control BRB and in early distal segments of alkalotic animals, but in late distal tubule a significant component of 0.14±0.033 nmol/cm² · sec was observed, which, however, is small when compared to the reabsorptive flow found in control Wistar rats, of 0.95±0.10 nmol/cm² · sec. In addition, 5×10^–4 m SITS had no effect on distal bicarbonate reabsorption in controls as well as on secretion in alkalotic Wistar and Brattleboro rats, which is compatible with the absence of effect of this drug on the apical Cl^–/HCO ₃ ^– exchange in other tissues. It is concluded that most distal alkalinization is not Cl^– dependent, and that Cl^–/HCO ₃ ^– exchange may be found in cortical distal tubule, but its magnitude is, even in alkalosis, markedly smaller than the reabsorptive flux, which predominates in the rats studied in this paper, keeping luminal pH lower than that of blood.     

Differential regulation of Na+/H+ exchange and H+ -ATPase by pH and HCO 3 − in kidney proximal tubules

This study examines the effects of acute in vitro acid-base disorders on Na⁺/H⁺ and H⁺-ATPase transporters in rabbit kidney proximal tubules (PT). PT suspensions were incubated in solutions with varying acid base conditions for 45 min and utilized for brush border membrane (BBM) vesicles preparation. BBM vesicles were studied for Na⁺/H⁺ exchange activity (assayed by ²²Na⁺ influx) or abundance (using NHE-3 specific antibody) and H⁺-ATPase transporter abundance (using antibody against the 31 kDa subunit). The Na⁺/ H⁺ exchanger activity increased by 55% in metabolic acidosis (pH 6.5, HCO ₃ ^– 3 mm) and decreased by 41% in metabolic alkalosis (pH 8.0, HCO ₃ ^– 90 mm). The abundance of NHE-3 remained constant in acidic, control, and alkalotic groups. H⁺-ATPase abundance, however, decreased in metabolic acidosis and increased in metabolic alkalosis by 57% and 42%, respectively. In PT suspensions incubated in isohydric conditions (pH 7.4), Na⁺/H⁺ exchanger activity increased by 29% in high HCO ₃ ^– group (HCO ₃ ^– 96 mm) and decreased by 16% in the low HCO ₃ ^– groups (HCO ₃ ^– 7mm. The NHE-3 abundance remained constant in high, normal, and low [HCO ₃ ^– ] tubules. The abundance of H⁺-ATPase, however, increased by 82% in high [HCO ₃ ^– ] and decreased by 77% in the low [HCO ₃ ^– ] tubules. In PT suspensions incubated in varying pCO₂ and constant [HCO ₃ ^– ], Na⁺/H⁺ exchanger activity increased by 35% in high pCO₂ (20% pCO₂, respiratory acidosis) and decreased by 32% in low pCO₂ (1.5% pCO₂, respiratory alkalosis) tubules. The NHE-3 abundance remained unchanged in high, normal, and low pCO₂ tubules. However, the H⁺-ATPase abundance increased by 74% in high pCO₂ and decreased by 69% in low pCO₂ tubules.The results of these studies suggest that the luminal Na⁺/H⁺ exchanger is predominantly regulated by pH whereas H⁺-ATPase is mainly regulated by [HCO ₃ ^– ] and/ or pCO₂. They further suggest that the adaptive changes in H⁺-ATPase transporter are likely mediated via endocytic/exocytic pathway whereas the adaptive changes in Na⁺/H⁺ exchanger are via the nonendocytic/exocytic pathway.The excellent technical assistance of Yollanda J. Hattabaugh, Gwen L. Bizal, and L. Yang is greatly appreciated. Portions of these studies were presented at the annual meeting of the American Society of Nephrology, Boston, MA, November 1993, and published in abstract form (J.Am.Soc.Neph. 4:840A, 1993)These studies were supported by a Merit Review Grant from the Department of Veterans Affairs and a grant-in-aid from the American Heart Association (to M.S.), a Baxter Health Care Grant (to B.B.), and the National Institute of Health Grants DK 38510 (to E.B.C. and M.C.R.) and DK 42086 (to E.B.C.).     

Effects of NH4 +, NO3 − and HCO3 − on apoplast pH in the outer cortex of root zones of maize, as measured by the fluorescence ratio of fluorescein boronic acid

A fluorimetric ratio technique was elaborated to measure apoplastic pH in the outer root cortex of maize (Zea mays L.) grown hydroponically. A newly synthesized fluorescent probe, fluorescein boronic acid (pK_a = 5.48), which covalently binds to the cell wall of the outer cell layers, was used. Under conditions of saturating ion concentrations the apoplastic pH was determined along the root axis ranging from 1 to 30 mm behind the root tip. Apoplastic pH was recorded for root segment areas (1 mm²), and pH values of high statistical significance were obtained. With an external solution of pH 5, the apoplastic pH was about pH 5.1 in the division zone, between pH 4.8 and 4.9 in the elongation region and about pH 4.9 in the root hair zone. At an external pH of 8.6, the difference between the external pH and the apoplastic pH was considerably more, with a pH of 5.2–5.3 in all root zones. Addition of 1 mM NH₄ ⁺ caused a small apoplastic pH decrease (0.05 of a pH unit) in all root zones. Apoplastic alkalization upon application of 6 mM NO₃ ⁻ was highest (0.3 of a pH unit) in the zone where root hairs emerge; in the division and early elongation zones, apoplastic pH increased only transiently. In the presence of 10 mM HCO₃ ⁻, NO₃ ⁻ elicited a higher and persistent alkalization (0.06–0.25 of a pH unit) in all root zones. Application of fusicoccin reduced apoplastic pH from 4.85 to 4.75 in the elongation zone, while inhibition of the H⁺-ATPase with vanadate alkalized the apoplast in the root hair zone from pH 5.4 to 5.6. The observed pH differences along the root axis upon differential N supply and application of HCO₃ ⁻ provide evidence that this new pH technique is a useful tool with which to measure apoplastic pH, and in future may permit measurements at microsites at the cell level by use of microscope imaging. Received: 26 August 1998 / Accepted: 4 May 1999     

Is CFTR an exchanger?: Regulation of HCO3−Transport and extracellular pH by CFTR

The disease, cystic fibrosis, is caused by the malfunction of the cystic fibrosis transmembrane conductance regulator. Expression of functional CFTR may normally regulate extracellular pH via control of bicarbonate efflux. Reports also suggest that the CFTR may be a Cl-/HCO3- exchanger. If true, this could be very important for treatment of CF given the airway host defense system is quite sensitive to pH, and acidic pH been found to increase mucus viscosity. We compared evidentiary support of four possible models of CFTR's role in the transport of bicarbonate: 1) CFTR as a Cl-channel that permits bicarbonate conductance, 2) CFTR as an anion Cl-/HCO3- exchanger (AE), 3.) CFTR as both a Cl-channel and an AE, and 4.) CFTR as a Cl-channel that allows for transport of bicarbonate and regulates an independent AE. The effect of stimulators and inhibitors of CFTR and AEs were evaluated via iodide efflux and studies of extracellular pH. This data, as well as that published by others, suggest that while CFTR may support and regulate bicarbonate flux it is unlikely it directly performs Cl-/HCO3- anion exchange.     

Evidence for involvement of protein kinase C in regulation of intracellular pH by Cl−/HCO 3 − antiport

Summary The activity of the main base-extruding mechanism in Vero cells, the Na⁺-independent Cl^–/HCO ₃ ^– antiport, increases 5- to 10-fold when the cytosolic pH (pH _i ) is increased over a narrow range close to neutrality. We have studied the effect on this regulation of stimulation and inhibition of protein kinase C by short-term and long-term treatment with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). After short-term treatment with TPA to stimulate the kinase, the threshold value for activation of the antiport is shifted to a more acidic pH. After prolonged treatment with TPA to downregulate protein kinase C the sensitivity of the antiport to variation in proton concentration was lowered, possibly by reducing the number of essential protonbinding sites. Concomitantly, the steady state pH _i of the cells was increased. The data indicate that protein kinase C is involved in the regulation of the Na⁺-independent Cl^–/HCO ₃ ^– antiport.     

Effect of pH and Glucose on the Stability of α-lactalbumin

The objective of this study is to understand the influence of pH and effect of cosolvent (glucose) on the stabilization of bovine α-lactalbumin by using ultrasonic techniques. Values of density, ultrasonic velocity and viscosity were measured for bovine α-lactalbumin (5 mg/ml) dissolved in phosphate buffer (pH 2, 5, 7, 9 and 12) solutions mixed with and without the cosolvent at 30 °C. These measurements were used to calculate few thermo-acoustical parameters such as adiabatic compressibility, intermolecular free length, acoustic impedance, relaxation time, relative association constant, the partial apparent specific volume and the partial apparent specific adiabatic compressibility for the said systems. The obtained results revealed a strong comparison between the effects of acidic and alkaline pH values on protein denaturation, i.e., the acidic pH are instantaneous and are of less magnitude whereas alkaline pH are slower but sharper. Further the present study supports the fact that the presence of glucose stabilizes α-lactalbumin against denaturation due to pH variation, which may be due to the strengthening of non-covalent interactions and the steric exclusion effect.     

Stimulation by HCO 3 − of Na+ transport in rabbit gallbladder

Summary Bicarbonate presence in the bathing media doubles Na⁺ and fluid transepithelial transport and in parallel significantly increases Na⁺ and Cl^– intracellular concentrations and contents, decreases K⁺ cell concentration without changing its amount, and causes a large cell swelling. Na⁺ and Cl^– lumen-to-cell influxes are significantly enhanced, Na⁺ more so than Cl^–. The stimulation does not raise any immediate change in luminal membrane potential and cannot be due to a HCO ₃ ^– -ATPase in the brush border. The stimulation goes together with a large increase in a Na⁺-dependent H⁺ secretion into the lumen. All of these data suggests that HCO ₃ ^– both activates Na⁺–Cl^– cotransport and H⁺–Na⁺ countertransport at the luminal barrier.Thiocyanate inhibits Na⁺ and fluid transepithelial transport without affecting H⁺ secretion and HCO ₃ ^– -dependent Na⁺ influx. It reduces Na⁺ and Cl^– concentrations and contents, increases the same parameters for K⁺, causes a cell shrinking, and abolishes the lumen-to-cell Cl^– influx. It enters the cell and is accumulated in the cytoplasm with a process which is Na⁺-dependent and HCO ₃ ^– -activated. Thus, SCN^– is likely to compete for the Cl^– site on the cotransport carrier and to be slowly transferred by the cotransport system itself.     

Effects of salicylate on HCO 3 − /Cl− exchange across the human erythrocyte membrane

Summary Changes in extracellular pH (pH _o ) in human red cell suspensions were monitored in a stopped-flow rapid reaction apparatus. A 20% suspension of washed human RBC in saline at pH 7 containing NaHCO₃ and extracellular carbonic anhydrase was mixed with an equal volume of buffered saline solution at pH 6.7. Sodium salicylate, when present, was added to both the erythrocyte suspension and the buffer solution. The effects of salicylate in the therapeutic to toxic concentration range on HCO ₃ ^– /Cl^– exchange were studied at 37°C. HCO ₃ ^– /Cl^– exchange flux was estimated using the extracellular buffer capacity and the difference betweendpH _o /dt using a control RBC suspension and that using a suspension of RBC whose anion exchange pathway was markedly inhibited. The results show that salicylate competitively decreases the rate of HCO ₃ ^– /Cl^– exchange, with inhibition increasing as salicylate concentration increases.K _I is 2.4mm. At a salicylate concentration of 10mm, HCO ₃ ^– /Cl^– exchange under the conditions of our experiments was inhibited by more than 70%. These findings are consistent with the possibility that CO₂ transfer in capillary bedsin vivo may be diminished in the presence of salicylate due to slowing of red cell HCO ₃ ^– /Cl^– exchange.     

Calcium ionophore-induced changes in HCO3− secretion and Cl− absorption in turtle bladder: relation to action of 3-isobutyl-1-methylxanthine

The calcium ionophore A23187 stimulates luminal alkalinization and inhibits Cl⁻ absorption in short-circuited urinary bladders of postprandial or alkalotic turtles. The ionophore appears to mimic the action of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) by its similar effects on HCO₃⁻ secretion and Cl⁻ absorption and by increasing cytosolic cAMP levels of isolated bladder epithelial cells. However, only A23187 (or ionomycin), but not IMBX or cAMP, elevated cytosolic Ca²⁺ of aequorin- or quin2-loaded cells. Since A23187, but not IBMX or cAMP inhibits luminal acidification, we postulate that cytosolic Ca²⁺ (1) regulates the acidification process by a cAMP-independent mechanism and (2) controls HCO₃⁻ secretion as well as Cl⁻ absorption, at least in part, via cAMP-mediated pathways.     

HCO3 − Transport in a Mathematical Model of the Pancreatic Ductal Epithelium

We have used computer modeling to investigate how pancreatic duct cells can secrete a fluid containing near isotonic (∼140 mm) NaHCO₃. Experimental data suggest that NaHCO₃ secretion occurs in three steps: (i) accumulation of HCO⁻ ₃ across the basolateral membrane of the duct cell by Na(HCO₃) _n cotransporters, Na⁺/H⁺ exchangers and proton pumps; (ii) secretion of HCO⁻ ₃ across the luminal membrane on Cl⁻/HCO⁻ ₃ antiporters operating in parallel with Cl⁻ channels; and (iii) diffusion of Na⁺ through the paracellular pathway. Programming the currently available experimental data into our computer model shows that this mechanism for HCO⁻ ₃ secretion is deficient in one important respect. While it can produce a relatively large volume of a HCO⁻ ₃-rich fluid, it can only raise the luminal HCO⁻ ₃ concentration up to about 70 mm. To achieve secretion of 140 mm NaHCO₃ by the model it is necessary to: (i) reduce the conductive Cl⁻ permeability and increase the conductive HCO⁻ ₃ permeability of the luminal membrane of the duct cell, and (ii) reduce the activity of the luminal Cl⁻/HCO⁻ ₃ antiporters. Under these conditions most of the HCO⁻ ₃ is secreted via a conductive pathway. Based on our data, we propose that HCO⁻ ₃ secretion occurs mainly by the antiporter in duct segments near the acini (luminal HCO⁻ ₃ concentration up to ∼70 mm), but mainly via channels further down the ductal tree (raising luminal HCO⁻ ₃ to ∼140 mm). Received: 15 November 1999/Revised: 29 March 2000     

Effect of β-carotene on catechol-induced genotoxicity in vitro: Evidence of both enhanced and reduced DNA damage

Abstract

Intake of antioxidants from the diet has been recognized to have beneficial health effects, but the potential benefit of taking antioxidants such as β-carotene as supplements is controversial. The aim of the present study was to evaluate the potential protective effects of a physiologically relevant concentration (2 μM) of β-carotene on the DNA damaging effects of catechol in mouse lymphoma L5178Y cells. Two different exposure protocols were used: simultaneous exposure to β-carotene and catechol for 3 h; and exposure to catechol for 3 h after 18 h pre-treatment with the vitamin. DNA damage was evaluated using the comet assay (employing one procedure for general damage, and another procedure, which also included oxidative DNA damage). Independent of exposure protocol and procedure for comet assay, β-carotene did not increase the basal level of DNA damage. However, at the highest concentration of catechol (1 mM), β-carotene was found to clearly increase the level of catechol-induced DNA damage, especially in the pre-treated cells. Interestingly, an opposite effect was observed at lower concentrations of catechol, but the β-carotene related reduction of catechol-induced genotoxicity was significant (P < 0.05) only for the procedure including oxidative damage induced by 0.5 mM catechol. Taken together our results indicate that β- carotene can both reduce and enhance the DNA damaging effects of a genotoxic agent such as catechol. This indicates that it is the level of catechol-induced DNA damage that seems to determine whether β-carotene should be regarded as a beneficial or detrimental agent when it comes to its use as a dietary supplement.     

Characterisation of CO2 and HCO3 − uptake in the cyanobacterium Synechocystis sp. PCC6803

The availability of a complete genome database for the cyanobacterium Synechocystissp. PCC6803 (glucose-tolerant strain) has raised expectations that this organism would become a reference strain for work aimed at understanding the CO₂-concentrating mechanism (CCM) in cyanobacteria. However, the amount of physiological data available has been relatively limited. In this report we provide data on the relative contributions of net HCO₃ ⁻ uptake and CO₂ uptake under steady state photosynthetic conditions. Cells were compared after growth at high CO₂ (2% v/v in air) or limiting CO₂ conditions (20 ppm CO₂). Synechocystishas a very high dependence on net HCO₃ ⁻ uptake at low to medium concentrations of inorganic carbon (Ci). At high Ci concentrations net CO₂ uptake became more important but did not contribute more than 40% to the rate of photosynthetic O₂ evolution. The data also confirm that high Ci cells of Synechocystissp. PCC6803 possess a strong capacity for net HCO₃ ⁻ uptake under steady state photosynthetic conditions. Time course experiments show that induction of maximal Ci uptake capacity on a shift from high CO₂ to low CO₂ conditions was near completion by four hours. By contrast, relaxation of the induced state on return of cells to high CO₂, takes in excess of 230 h. Experiments were conducted to determine if Synechocystissp. PCC6803 is able to exhibit a `fast induction' response under severe Ci limitation and whether glucose was capable of causing a rapid inactivation in Ci uptake capacity. Clear evidence for either response was not found. This revised version was published online in August 2006 with corrections to the Cover Date.