pH regulation in HT29 colon carcinoma cells

The pH regulation in HT₂₉ colon carcinoma cells has been investigated using the pH-sensitive fluorescent indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Under control conditions, intracellular pH (pH_i) was 7.21±0.07 (n=22) in HCO ₃ ^– -containing and 7.21±0.09 (n=12) in HCO ₃ ^– -free solution. HOE-694 (10 mol/l), a potent inhibitor of the Na⁺/H⁺ exchanger, did not affect control pH_i. As a means to acidify cells we used the NH ₄ ⁺ /NH₃ (20 mmol/l) prepulse technique. The mean peak acidification was 0.37±0.07 pH units (n=6). In HCC ₃ ^– -free solutions recovery from acid load was completely blocked by HOE-694 (1 mol/l), whereas in HCO3 ₃ ^– -containing solutions a combination of HOE-694 and 4,4-diisothiocyanatostilbene-2, 2-disulphonate (DIDS, 0.5 mmol/l) was necessary to show the same effect. Recovery from acid load was Na⁺-dependent in HCO ₃ ^– -containing and HCO ₃ ^– -free solutions. Removal of external Cl^– caused a rapid, DIDS-blockable alkalinization of 0.33±0.03 pH units (n=15) and of 0.20±0.006 pH units (n=5), when external Na⁺ was removed together with Cl^–. This alkalinization was faster in HCO ₃ ^– -containing than in HCO ₃ ^– -free solutions. The present observations demonstrate three distinct mechanisms of pH regulation in HT₂₉ cells: (a) a Na⁺/H⁺ exchanger, (b) a HCO ₃ ^– /Cl^– exchanger and (c) a Na⁺-dependent HCC ₃ ^– transporter, probably the Na⁺-HCO ₃ ^– /Cl^– antiporter. Under HCO ₃ ^– — free conditions the Na⁺/H⁺ exchanger fully accounts for recovery from acid load, whereas in HCO ₃ ^– -containing solutions this is accomplished by the Na⁺/H⁺ exchanger and a Na⁺-dependent mechanism, which imports HCO ₃ ^– . Recovery from alkaline load is caused by the HCO ₃ ^– /Cl^– exchanger.This study was supported by DFG Gr 480/10     

HCO 3 − -dependent ACh-activated Na+ influx in sheep parotid secretory endpieces

In the present study we used the Na⁺-sensitive fluorescent dye SBFI and optical measurement of endpiece volume to investigate the transport of Na⁺ in sheep parotid secretory cells. Sheep parotid endpiece cells bathed in a HCO ₃ ^– -free Cl^–-rich solution had a resting intracellular Na⁺ concentration ([Na⁺]_i) of 17±2 mmol/l (n=39). Exposure of the cells to a 2-min pulse of acetylcholine (ACh) (3×10^–7 mol/l) in a HCO ₃ ^– -free bathing solution produced no change in [Na⁺]_i or in cell volume. Changing from a Cl^–-containing HCO ₃ ^– -free bath solution to a Cl^– solution containing 25 mmol/l HCO ₃ ^– caused the endpieces to swell by 8±2 % (n=11) and the [Na⁺]_i to increase by 10±2 mmol/l (n=14). Subsequent exposure of the cells to ACh led to shrinkage of the cells by 12±2 % from the volume in the HCO ₃ ^– -containing solution prior to ACh exposure, with the maximum decrease occurring after 29±7 s (n=9). This shrinkage was accompanied by a rapid and transient increase in [Na⁺]_i, the [Na⁺]_i reaching a peak at 70±5 mmol/l above the unstimulated level (n=9). Substitution of gluconate for Cl^– did not significantly alter the effects of HCO ₃ ^– on unstimulated [Na⁺]_i or endpiece volume, nor did it significantly inhibit the effects of ACh on these two parameters when HCO ₃ ^– was present. Addition of 200 mol/l dihydrogen-4,4-diisothiocyanatostilbene-2,2-disulfonic acid (H₂-DIDS) to the gluconate/HCO ₃ ^– solution significantly reduced the peak of the ACh-induced increase in [Na⁺]_i to 34±10 mmol/l (n=4), but did not have any significant effect on the magnitude of the ACh-induced shrinkage. At 500 mol/l, H₂-DIDS abolished the ACh-induced increase in [Na⁺]_i and also significantly reduced the shrinkage due to ACh. Finally, we found that the rate of endpiece shrinkage following ACh stimulation did not depend on the presence of Cl^–.We interpret these results as indicating that sheep parotid secretory cells do not contain significant Na⁺-K⁺-2Cl^– co-transport activity and do not actively accumulate Cl^–. Rather, the mechanism of spontaneous basal secretion by these cells, in the presence of extracellular HCO ₃ ^– , is based on the accumulation of HCO ₃ ^– by the Na⁺-H⁺ exchanger. During ACh stimulation, the concentration of HCO ₃ ^– in the cytosol is also maintained by the operation of a H₂-DIDS-sensitive Na⁺-HCO ₃ ^– co-transporter. HCO ₃ ^– efflux across the apical membrane occurs via a HCO ₃ ^– conductance pathway rather than by the coupled operation of a Cl^– channel and a Cl^–-HCO ₃ ^– exchanger.     

Evidence for Na+ dependent rheogenic HCO 3 − transport in fused cells of frog distal tubules

The mechanism of HCO ₃ ^– transport was studied applying microelectrodes in giant cells fused from single epithelial cells of the diluting segment of frog kidney. A sudeen increase of extracellular HCO ₃ ^– concentration from 10 to 20 mmol/l at constant pH hyperpolarized the cell membrane potential of the fused cell. This cell-voltage response was totally abolished by 10^–3 mol/l SITS and significantly reduced by 10^–4 mol/l acetazolamide or by omission of Na⁺ from the extracellular perfusate. Removal of Na⁺ from the perfusate caused a transient depolarization. Reapplication of Na⁺ induced a transient hyperpolarization. 10^–3 mol/l SITS abolished the cell-voltage response to removal and reapplication of Na⁺. In the intact diluting segment of the isolated perfused frog kidney peritubular perfusion of 10^–4 mol/l acetazolamide reduced the limiting transepithelial electrochemical gradient for H⁺ significantly from 30±4 mV to 14±3 mV. The results suggest: (i) In the diluting segment of the frog kidney a Na⁺-dependent rheogenic HCO ₃ ^– transport system exists across the peritubular cell membrane. (ii) This rheogenic peritubular Na⁺/HCO ₃ ^– cotransporter cooperates with a Na⁺/H⁺ exchanger in the luminal membrane, thus driving HCO ₃ ^– reabsorption. (iii) Reabsorption of HCO ₃ ^– and secretion of H⁺ depend upon the presence of carbonic anhydrase.     

Electrophysiological study of transport systems in isolated perfused pancreatic ducts: properties of the basolateral membrane

In order to study the mechanism of pancreatic HCO ₃ ^– transport, a perfused preparation of isolated intra-and interlobular ducts (i.d. 20–40 m) of rat pancreas was developed. Responses of the epithelium to changes in the bath ionic concentration and to addition of transport inhibitors was monitored by electrophysiological techniques. In this report some properties of the basolateral membrane of pancreatic duct cells are described. The transepithelial potential difference (PD_te) in ducts bathed in HCO ₃ ^– -free and HCO ₃ ^– -containing solution was –0.8 and –2.6 mV, respectively. The equivalent short circuit current (I_sc) under similar conditions was 26 and 50 A·cm^–2. The specific transepithelial resistance (R_te) was 88 cm². In control solutions the PD across the basolateral membrane (PD_bl) was –63±1 mV (n=314). Ouabain (3 mmol/l) depolarized PD_bl by 4.8±1.1 mV (n=6) within less than 10 s. When the bath K⁺ concentration was increased from 5 to 20 mmol/l, PD_bl depolarized by 15.9±0.9 mV (n=50). The same K⁺ concentration step had no effect on PD_bl if the ducts were exposed to Ba²⁺, a K⁺ channel blocker. Application of Ba²⁺ (1 mmol/l) alone depolarized PD_bl by 26.4±1.4 mV (n=19), while another K⁺ channel blocker TEA⁺ (50 mmol/l) depolarized PD_bl only by 7.7±2.0 mV (n=9). Addition of amiloride (1 mmol/l) to the bath caused 3–4 mV depolarization of PD_bl. Furosemide (0.1 mmol/l) and SITS (0.1 mmol/l) had no effect on PD_bl. An increase in the bath HCO ₃ ^– concentration from 0 to 25 mmol/l produced fast and sustained depolarization of PD_bl by 8.5±1.0 mV (n=149). It was investigated whether the effect of HCO ₃ ^– was due to a Na⁺⁺-dependent transport mechanism on the basolateral membrane, where the ion complex transferred into the cell would be positively charged, or whether it was due to decreased K⁺ conductance caused by lowered intracellular pH. Experiments showed that the HCO ₃ ^– effect was present even when the bath Na⁺ concentration was reduced to a nominal value of 0 mmol/l. Similarly, the HCO ₃ ^– effect remained unchanged after Ba²⁺ (5 mmol/l) was added to the bath. The results indicate that on the basolateral membrane of duct cells there is a ouabain sensitive (Na⁺+K⁺)-ATPase, a Ba²⁺ sensitive K⁺ conductance and an amiloride sensitive Na⁺/H⁺ antiport. The HCO ₃ ^– effect on PD_bl is most likely due to rheogenic anion exit across the luminal membrane.     

Uncoupling of Na+H+ from Cl−HCO 3 − exchange under some steady state conditions in rabbit gallbladder

The transapical Cl^– influx and transepithelial Na⁺ transport were measured in rabbit gallbladder. Only 11.7% of the transported Na⁺ was found to be accompanied by HCO ₃ ^– . 10^–4 M SITS eliminated the HCO ₃ ^– dependent fraction of Cl^– influx (50%) but did not significantly alter intracellular Na⁺ activity and Na⁺ transport. Exposure to HCO₃-free salines or to 10^–4 M acetazolamide about halved Cl^– influx and Na⁺ transport. 25 mM SCN^– reduced Cl^– influx to zero, decreased intracellular Na⁺ activity, but only halved Na⁺ transport which under these conditions was abolished only in the absence of HCO ₃ ^– . Exposure to a Cl^–-free saline produced effects similar to those caused by SCN^–. These resuits suggest that when Cl^–/HCO ₃ ^– exchange is inhibited at the apical membrane, Na⁺/H⁺ exchange and transepithelial Na⁺ transfer are unmodified if HCO ₃ ^– is available for transport. The permanent uncoupling of the exchangers and the elevated transepithelial transport of Na⁺ are not due to an increased activity of the parallel Na⁺–Cl^– cotransport but to a redirection of HCO ₃ ^– flux toward the basolateral side.     

Regulation of intracellular pH in cultured bovine retinal pigment epithelial cells

Regulation of intracellular pH (pH_i) in bovine retinal pigment epithelium (RPE) was investigated in cell culture. pH_i was measured using the pH-sensitive absorbance of intracellularly trapped 5 (and 6)-carboxy-dimethyl-fluorescein (CDMF). (1) Regulation of pH_i after induction of an acid load by removal of NH₄Cl could be blocked either totally by removal of extracellular sodium, or subtotally (about 90%) by application of amiloride (1 mmol/l). Additional flux measurements revealed a dose-dependent, amiloride-sensitive²²Na⁺-uptake into Na⁺-loaded cells. Both results suggest the presence of a Na⁺/H⁺ antiport.(2) When alkalinization of the cells was induced by preincubation with 50 mmol/l acetate in HCO ₃ ^– -Ringer's and subsequent removal of the weak acid, the following regulation was dependent on the presence of extracellular chloride. This process could be blocked with DIDS (1 mmol/l), suggesting the presence of a Cl^–/HCO ₃ ^– exchange mechanism.(3) We found no evidence for a Na⁺/HCO ₃ ^– -cotransport, which had been postulated to be present in RPE by others. We conclude that two processes are involved in regulation of pH_i in RPE: A Na⁺/H⁺ antiport responsible for recovery of pH_i from acid load, and a DIDS-sensitive Cl^–/HCO ₃ ^– exchange mechanism responsible for recovery of pH_i after alkalinization.Parts of this work jhave been published in abstract from [20, 21]     

Thrombin-induced cytosolic alkalinization in human platelets occurs without an apparent involvement of HCO 3 − /Cl− exchange

We have estimated the changes in cytosolic pH (pH_i) that occur when human platelets are stimulated by thrombin. Changes in pH_i were estimated (i) from the H⁺ efflux across the plasma membrane using an extracellular pH electrode and (ii) using an intracellular pH-sensitive fluorescent dye (BCECF). Stimulation of platelets with thrombin (0.5 unit/ml) resulted in an H⁺ efflux that averaged 7.7±1.6 mol/10¹¹ platelets (means±SD) leading to an increase in pH_i, from 7.05±0.04 to 7.45±0.05. Both H⁺ efflux and pH_i changes were unaffected by 0.1 mM 4,4-diisothiocyanostilbene-2,2 disulphonate (DIDS), 0.1 mM 4-acetamido 4-isothiostilbene-2,2-disulphonic acid (SITS), or 0.5 mM bumetanide, suggesting no involvement of anion transport systems, e.g. an HCO ₃ ^– /Cl^– exchange. Removal of HCO ₃ ^– or Cl^– from the suspending buffer had no effect on the extent of the rise in pH_i. After blockade of Na⁺/H⁺ exchange by 100 M ethylisopropylamiloride (EIPA), thrombin induced a decrease in pH_i the rate of which averaged 0.39 unit/min in HCO ₃ ^– -containing medium, and 0.57 unit/min in HCO ₃ ^– -free medium. The cytosolic buffer capacity for H⁺ was determined by the nigericin/ NH₄Cl technique in BCECF-loaded platelets and averaged 25.3 mmol/(1xpH) in buffer containing 8 mM HCO ₃ ^– , but only 17.2 mmol/(1xpH) in HCO ₃ ^– -free buffer. The total amount of H⁺ transferred by Na⁺/H⁺ exchange can be estimated from our measurements at 10 mmol/l platelet cytosol in the absence of HCO ₃ ^– and to 14 mmol/l platelet cytosol in the presence of HCO ₃ ^– , and is in good agreement with the estimated amount of Na⁺ uptake by ADP-stimulated platelets. We conclude that net extrusion of H⁺ from stimulated platelets is predominantly mediated by Na⁺/H⁺ exchange without an apparent contribution of HCO ₃ ^– /Cl^– exchange.     

Evidence for a Na+/H+ exchanger at the basolateral membranes of the isolated frog skin epithelium: Effect of amiloride analogues

We have investigated the possible existence of a Na⁺/H⁺ ion exchanger in the frog skin epithelium by using isotopic methods and two amiloride analogues: 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and phenamil. We found phenamil to be a specific blocker of sodium entry to its cellular transport compartment since it inhibited both the transepithelial Na⁺ influxes (J ₁₃) with aK _I of 4·10^–7 mol/l and the Na⁺ pool (control: 77±4 neq·h^–1·cm^–2; phenamil: 21±1 neq·h^–1·cm^–2). On the contrary EIPA (10^–5 mol/l) had no effect onJ ₁₃ nor on the apical Na⁺ conductance. Acidification of the epithelium by passing from a normal Ringer (25 mmol/l HCO ₃ ^– , 5% CO₂, pH 7.34) to a HCO ₃ ^– -free Ringer (5% CO₂, pH 6.20) while blocking the Na⁺ conductance with phenamil, produced a large stimulation of Na⁺ influxes exclusively across the basolateral membranes (J ₃₂), after return to a normal Ringer (J ₃₂=706±76 and 1635±199 neq·h^–1·cm^–2 in control and acid-loaded epithelia respectively). The stimulation ofJ ₃₂ was initiated when the epithelia were acid-loaded with Ringer of pH lower than 6.90 and was blocked by amiloride (K _I=7·10^–6 mol/l) and EIPA (K _I=5·10^–7 mol/l) whereas phenamil had no effect. In na⁺-loaded epithelia (ouabain treated) the Na⁺ efflux across the basolateral membranes was stimulated by an inwardly directed proton gradient and was blocked by EIPA (10^–5 mol/l) or amiloride (10^–4 mol/l), a result suggesting reversibility of the mechanism. We conclude that a Na⁺ permeability mediated by a Na⁺/H⁺ ion exchanger exists in the basolateral membranes, which is stimulated by intracellular acidification and is sensitive to amiloride or EIPA. This exchanger is proposed to be involved in intracellular pH regulation.     

Na+-HCO3 − cotransporter and intracellular pH regulation in chicken enterocytes

The current studies examine the presence of the Na⁺-HCO₃ ^– cotransporter in chicken enterocytes and its role in cytosolic pH (pH_i) regulation. The pH-sensitive dye 2,7-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF) was used to monitor pH_i. Under resting conditions, pH_i was 7.25 in solutions buffered with bis(2-hydroxyethyl)-1-piperazine ethanesulphonic acid (HEPES) and 7.17 in those buffered with HCO₃ ^–. Removal of external Na⁺ decreased pH_i and readdition of Na⁺ rapidly increased pH_i towards the control values. These Na⁺-dependent changes were greater in HCO ₃ ^– than in HEPES-buffered solutions. In HCO ₃ ^– - free solutions the Na⁺-dependent changes in pH_i were prevented by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and unaffected by 4,4-diisothiocyanatostilbene disulphonic acid (H₂-DIDS). In the presence of HCO ₃ ^– , the Na⁺-induced changes in pH_i were sensitive to both EIPA and H₂-DIDS. In the presence of EIPA, cells partially recovered from a moderate acid load only when both Na⁺ and HCO ₃ ^– were present. This pH_i recovery, which was EIPA resistant, and dependent on Na⁺ and HCO ₃ ^– , was inhibited by H₂-DIDS and occurred at equal rates in both Cl^–-containing and Cl^–-free solutions. Kinetic analysis of the rate of HCO ₃ ^– - and Na⁺- dependent pH_i recovery from an acid load as a function of the Na⁺ concentration revealed first-order kinetics with a Michaelis constant, K _m, of 11 mmol/l Na⁺. It is concluded that in HCO₃ ^/– buffered solutions both the Na⁺/H⁺ exchanger and the Na⁺-HCO₃ ^– cotransporter participate in setting the resting pH_i in isolated chicken enterocytes and help the recovery from acid loads.     

Transport properties of the basolateral membrane of the oxyntic cells in frog fundic gastric mucosa

The conductive properties of the basolateral membrane of oxyntic cells (OC) of frog fundic gastric mucosa were investigated by utilizing the microelectrode technique. By examining the response of the basolateral cell membrane potential difference,V _cs, to sudden ion concentration changes in the serosal bath it was concluded that the basolateral membrane of OC has a high Ba²⁺-sensitive K⁺-conductance, and no Cl^–-conductance both in resting (cimetidine) and in stimulated (histamine) state. The response ofV _cs to serosal Cl^–-removal, consisting in a slight hyperpolarization (anomalous Nernst response), could not be explained by possible permeability changes to K⁺ and Na⁺ since the potential response to Cl^– was essentially preserved by blocking K⁺-permeability with Ba²⁺ and replacing all Na⁺ by choline. Conversely, hyperpolarization ofV _cs after Cl^–-free perfusion was abolished by exposure to HCO ₃ ^– -free solution, indicating that HCO ₃ ^– -ions are required at the serosal bath for Cl^– to get his effect. It was investigated wether the effect of Cl^– was due to an electrogenic Na⁺(HCO ₃ ^– ) _n /Cl^– exchange mechanism on the basolateral membrane. Experiments showed that the potential response to HCO ₃ ^– -removal and to Na⁺-removal, consisting in a depolarization ofV _cs, was similar both in presence and in absence of Cl^–. Furosemide (0.5 mmol/l) had no effect on steadyV _cs andV _t. The electrophysiological analysis of the data led to excluding the involvement of Na-Cl, Na-2Cl and NaK-2Cl cotransports, and to including the existence of an electrogenic Na⁺(HCO ₃ ^– ) _n /Cl^– exchange process, while suggests the presence of an electroneutral Cl^–/HCO ₃ ^– exchange mechanism to explain Cl^–-transport across the basolateral membrane of OC.This work was supported by a research grant from Ministero della Pubblica Istruzione, Rome, Italy     

Mechanism of NaCl secretion in the rectal gland of spiny dogfish (Squalus acanthias)

Rectal gland tubules (RGT) of spiny dogfish were dissected and perfused in vitro. Transepithelial PD (PD_te), resistance (R_te), the PD across the basolateral membrane (PD_bl) and intracellular chloride and potassium activities (a _Cl– ^cell ,a _K+ ^cell ) were measured. In a first series, 67 RGT segments were perfused with symmetric shark Ringers solution. The bath perfusate contained in addition db-cAMP 10^–4, forskolin 10^–6, and adenosine 10^–4 mol · l^–1. PD_te was –11±1 (n=67) mV lumen negative, R_te 27±2 (n=47) cm². PD_bl –75±0.4 (n=260) mV.a _K+ ^cell anda _Cl– ^cell were 109±22 (n=4) and 38±4 (n=36) mmol · l^–1 respectively. These data indicate that Cl^– secretion across the RGT must be an uphill transport process, whereas secretion of Na⁺ could be driven by the lumen negative PD_te. Intracellular K⁺ is 14 mV above equilibrium with respect to the basolateral membrane PD and Cl^– is 23 mV above equilibrium across the apical membrane. In series 2, the conductivity properties of the apical and basolateral membrane as well as that of the paracellular pathway were examined in concentration step experiments. Decrease of the basolateral K⁺ concentration led to a rapid hyperpolarization of PD_bt with a mean slope of 19 mV per decade of K⁺ concentration change. Addition of 0.5 mmol · l^–1 Ba²⁺ to the bath solution lead to a marked depolarization and abolished the response to K⁺ concentration steps. In the lumen a Cl^– concentration downward step led to a depolarization of the lumen membrane; resulting in a mean slope of 18 mV per decade of Cl^– concentration change. When dilution potentials were generated across the epithelium, the polarity indicated that the paracellular pathway is cation selective. In series 3 the equivalent short circuit current (I_sc=PD_te/R_te) was determined as a function of symmetrical changes in Na⁺ concentration, with Cl^– held at 276 mmol · l^–1, and as a function of symmetrical changes in Cl^– concentration, with Na⁺ held at 278 mmol · l^–1 I_sc was a saturable function of Na⁺ concentration (Hill coefficient 0.9±0.1,K _1/2 4.4 mmol · l^–1,n=7) and also a saturable function of Cl^– concentration (Hill coefficient 2.0±0.1,K _1/2 75 mmol · l^–1,n=11). These data are compatible with the assumption that the carrier responsible for NaCl uptake has a 1 Na⁺ per 2 Cl^– stoichiometry. In series 4, the effect of a K⁺ concentration downward step on PD_bl anda _Cl– ^cell transients was followed with high time resolution in the presence and absence of basolateral furosemide (5 · 10^–5 to 10^–4 mol · l^–1) in an attempt to examine whether K⁺ reduction on the bath side inhibits Na⁺Cl^– uptake by the carrier system as does e.g. furosemide. The data indicate that removal of K⁺ from the bath side exerts an effect comparable to that of furosemide, i.e. it inhibits the carrier. We conclude that NaCl secretion in the RGT cell comprises at the least the following components: In the basolateral membrane, the (Na⁺+K⁺)-ATPase, probably the Na⁺ 2 Cl^–K⁺ carrier, and a K⁺ conductance. In the apical membrane a Cl^– conductance; and a Na⁺ conductive paracellular pathway.Supported by Deutsche Forschungsgemeinschaft DFG-Gr 480/8-1. Parts of this study have been presented at the 3rd International Symposium on Ion Selective Electrodes, Burg Rabenstein 1983, 16th Annual Meeting American Society of Nephrology, Washington DC 1983, 49th Tagung der Deutschen Physiologischen Gesellschaft, Dortmund 1984. A summary of the present study was published in Bulletin Mount Desert Island Biological Laboratory (Vol. 83)     

The mechanism of action of harmaline on renal solute transport

Summary The effect of the hallucinogenic drug harmaline was tested on rat kidney proximal tubular solute and water transport, using in vivo micropuncture and electrophysiological techniques as well as in vitro biochemical techniques. During peritubular application harmaline (5 mmol/l) was found to block net tubular volume absorption reversibly (by 85%) through inhibition of active Na⁺ transport and possibly active HCO ₃ ^– transport. The inhibition was accompanied by a rapid strong depolarization of the tubular cell membranes. As a biochemical equivalent harmaline inhibited the Na⁺–K⁺-ATPase and the Mg²⁺-ATPase of peritubular cell membrane fractions as well as the HCO ₃ ^– -stimulated ATPase of a brush border membrane fraction with similar kinetics. By studying glucose tracer efflux and by measuring cell membrane potential and conductance changes in response to glucose perfusions, no evidence for a direct effect of harmaline on Na⁺-glucose (or amino acid) cotransport mechanisms in the brush border could be obtained. The data suggest that harmaline does not specifically compete with Na⁺ for transport sites. Neither are the cotransport systems in the brush border membrane specifically inhibited, nor could the inhibition of the Na⁺ pump in the peritubular cell membrane simply result from a competition between harmaline and Na⁺.     

Bicarbonate reabsorption in the papillary collecting duct of rats

Using the technique of capillary perfusion and simultaneous luminal stop flow microperfusion the reabsorption of bicarbonate and glycodiazine from the papillary collecting duct was evaluated. Starting with equal H¹⁴CO ₃ ^– and³H-glycodiazine concentrations in the luminal and peritubular perfusates, the decrease in the luminal concentration at 10 and 45 s contact time was measured.In control rats with 25 mmol/l HCO ₃ ^– in the perfusates the rate of HCO ₃ ^– reabsorption calculated from the 10 s values was 0.34 nmol cm^–2s^–1. In acute metabolic acidosis, the rate of bicarbonate reabsorption was 2,3 times higher. In metabolic alkalosis, the rate of bicarbonate absorption dropped to 13% of the control values. Also the 45 s values of acidotic and alkalotic animals differed significantly from each other. With 25 mmol/l glycodiazine in both perfusates the rate of biffer reabsorption as calculated from the 10 s values was 0.76 nmol cm^–2s^–1 in control rats and did not deviate significantly from this value in acidotic and alkalotic animals.In control rats the bicarbonate reabsorption in % was the same, no matter whether both luminal and capillary perfusate contained 25 mmol/l bicarbonate or 10 mmol/l. In acidotic rats the rate of HCO ₃ ^– reabsorption did not change significantly if all Na⁺ in the perfusates was replaced by choline (0.88 versus 0.79 nmol cm^–2s^–1 at 25 mmol/l HCO ₃ ^– ). When in acidotic rats 0.1 mmol/l acetazolamide or 1 mmol/l SITS (4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid) was added to both perfusates the rate of HCO ₃ ^– reabsorption dropped by 75 and 58%, respectively. A potassium deficient diet for one week and DOCA administration had no influence on the bicarbonate reabsorption of rats which were on standard diet.The data indicate that (1) the buffer reabsorption from the papillary collecting duct is rather due to H⁺ ion secretion than to buffer anion reabsorption. (2) The adaptation to metabolic acidosis and alkalosis is specific for bicarbonate and not seen with glycodiazine. (3) Within the concentration range tested the HCO ₃ ^– reabsorption rises linearly with the HCO ₃ ^t- concentration. (4) The HCO ₃ ^– reabsorption in the papillary collecting duct is Na⁺-independent, it can be inhibited by acetazolamide and SITS, but is not influenced by K⁺-deficient diet plus DOCA.     

Pathways of phosphate transport in chick jejunum

The effect of vitamin D₃ on intestinal phosphate (P_i) absorption was studied in everted sacs prepared from jejunum of either vitamin D-deficient (–D) or vitamin D-replete (+D) chicks. Vitamin D₃ stimulates the maximal velocity (V _max) of a mucosal active P_i transport mechanism from 125 to 314 nmol·min^–1·g^–1 tissue.K _m of this process remains virtually unchanged (–D: 0.15 mmol·l^–1; + D: 0.18 mmol·l^–1).Active P_i entry into the epithelium depends on extracellular Na⁺. Reduction of buffer Na⁺ reducesV _max in the + D group to 182 nmol·min^–1·g^–1 tissue but has no significant effect in the –D animals (V _max=105 nmol·min^–1·g^–1 tissue). In this group, the predominant effect of Na⁺ substitution is a shift ofK _m to 1.13 mmol·l^–1, whileK _m in the +D group is changed only to 0.53 mmol·l^–1.Transeptithelial P_i transport in the + D group involves the mucosal phosphate pump and hence an intracellular pathway, proceeding at a rate of 48 nmol·min^–1·g^–1 tissue. This is in contrast to –D P_i transfer (8 nmol·l^–1·g^–1 tissue) which is by a diffusional, Na⁺-insensitive, and presumably paracellular pathway.Transepithelial calcium transport (–D: 3.3 nmol·min^–1·g^–1; + D: 7.6 nmol·min^–1·g^–1 tissue) does not require the presence of extracellular Na⁺ and apparently involves pathways different from those of the P_i absorptive system.Presented in part at the Annual Meeting of the Austrian Biochemical Society, Salzburg, September 1978     

Die Bedeutung der HCO 3 − -ATPase in der H+/HCO 3 − -Sekretion

Zusammenfassung Dem aktiven Puffertransport, der an Magen und Niere zur H⁺-Sekretion, am Pankreas und Speicheldrüse zur HCO ₃ ^– -Sekretion führt, ist eine ATP-Phosphohydrolase zugeordnet, die typischerweise durch HCO ₃ ^– -Ionen stimuliert wird. Im Gegensatz zur (Na⁺-K⁺)-ATPase, dem Transportenzym des Na⁺-Transportes, benötigt dieses Enzym nurein Ion und ist Ouabain unempfindlich. Die HCO ₃ ^– -ATPase ist an die Plasmamembran der einzelnen Epithelien gebunden, jedoch im Gegensatz zur (Na⁺-K⁺)-ATPase luminal lokalisiert. Das Enzym ändert seine Aktivität proportional zur Rate des aktiven Puffertransportes, was seine Bedeutung als Transportenzym unterstreicht. Es werden Krankheitsbilder des gestörten Puffertransportes aufgeziegt, bei denen möglicherweise der Defekt im HCO ₃ ^– -ATPase-System zu suchen ist.     

Stimulation of colonic anion secretion by monochloramine: action sites

During inflammatory bowel disease, reactive oxygen metabolites are released by phagocytes reacting with intraluminal NH₃ to produce monochloramine (NH₂Cl). NH₂Cl is assumed to play role in the pathogenesis of inflammation-associated diarrhoea, as it is able to induce intestinal secretion. The aim of the present study was to determine the action sites of NH₂Cl in rat colonic epithelium with Ussing chamber and fura-2 experiments. In intact mucosa, NH₂Cl (5·10^–6–10^–4 mol·l^–1) evoked a concentration-dependent increase in short-circuit current (I_sc), consistent with the induction of anion secretion, as demonstrated by anion substitution and transport blocker experiments. When the apical membrane was permeabilised by the ionophore nystatin, two basolateral action sites of NH₂Cl (5·10^–5 mol·l^–1) could be identified, i.e. an increase in the K⁺ conductance and a stimulation of the Na⁺–K⁺ pump. When tissues were basolaterally depolarised by a high K⁺ concentration, the stimulation of an apical Cl^– conductance by NH₂Cl was observed. In isolated colonic crypts loaded with the Ca²⁺-sensitive fluorescent dye fura-2, NH₂Cl (5·10^–5 mol·l^–1) evoked an increase in the intracellular Ca²⁺ concentration. This increase was independent from the presence of Ca²⁺ in the extracellular medium, but was inhibited by blockade of intracellular sarcoplasmatic, endoplasmatic Ca²⁺-ATPases with cyclopiazonic acid (10^–5 mol·l^–1). The NH₂Cl-evoked Ca²⁺ release was sensitive against inhibition of ryanodine receptors with ruthenium red (5·10^–5 mol·l^–1) and against inhibition of inositol-1,4,5-trisphosphate (IP₃) receptors with 2-aminoethoxydiphenylborate (10^–4 mol·l^–1). Both blockers also inhibited the NH₂Cl-induced increase in I_sc. These results indicate that an intracellular Ca²⁺ release via ryanodine and/or IP₃ receptors is involved in oxidant stimulation of anion secretion in rat colon.     

Hypertonicity in fused Madin-Darby canine kidney cells: transient rise in NaHCO3 followed by sustained KCl accumulation

We investigated mechanisms of regulatory volume increase in fused Madin-Darby canine kidney (MDCK) cells, a cell line originally derived from renal collecting duct. The intracellular ion concentrations as well as the concentration of the volume marker tetramethylammonium⁺ were measured by means of ion-selective microelectrodes. Application of hypertonic Ringer bicarbonate solution (+150 mmol/l mannitol) resulted in cell shrinkage to 84±2% of the initial cell volume (shrinkage expected for an ideal osmometer = 66%), indicating a significant regulatory volume increase. During the first 90 s of the hypertonic stress, a transient increase in intracellular Na⁺ and HCO ₃ ^– concentrations was observed. It was followed by a sustained increase in intracellular K⁺ and Cl^– concentrations. Ouabain (0.1 mmol/l) as well as amiloride (1 mmol/l) reduced K⁺ accumulation significantly, whereas the H⁺ /K⁺-ATPase inhibitor SCH 28080 had no effect. Hypertonic stress hyperpolarized the cell membrane potential by 19±2 mV, owing to the decrease of the ratio of Cl^– conductance to K⁺ conductance of the cell membrane. We conclude: (a) acute hypertonic stress activates Na⁺/H⁺ exchange in MDCK cells; (b) transient alteration of intracellular Na⁺ and pH stimulates Na⁺/K⁺-ATPase and Cl^–/HCO ₃ ^– exchange, both leading to the sustained intracellular accumulation of KCl; (c) a high intracellular KCl concentration is maintained by the partial reversion of the Cl^–/K⁺ conductance ratio of the plasma membrane.     

Requirement of HCO 3 − for Cl−-absorption in seawater-adapted eel intestine

The role of HCO ₃ ^– /CO₂ buffer in Cl^– absorption was examined in the in vitro perfused eel intestine adapted to seawater. Cl^– absorption, expressed as short/circuit current (I _sc), was measured in either 20 mM HCO ₃ ^– /1% CO₂ Ringer or HEPES Ringer, pH 8.0. Unilateral (mucosal or serosal) substitution of HCO ₃ ^– /CO₂ with HEPES/O₂ was without effect on I _sc and transepithelial voltage (V _t), whereas bilateral removal of HCO ₃ ^– /CO₂ reduced I _sc and V _t by 50%, indicating that the presence of HCO ₃ ^– /CO₂ buffer at one side of the epithelium is sufficient to keep Cl^– absorption at the maximum rate. We examined in further detail the individual components of the HCO ₃ ^– /CO₂ system that stimulates Cl^– absorption. We found that, in tissues bathed with HEPES Ringer, addition of 1% CO₂ to the luminal or serosal solution (final pH=7.6 in the chamber) had no effect on I _sc and V _t, while both electrical parameters could be restored to control values by unilateral (luminal or serosal) substitution of HEPES Ringer with 20 mM HCO ₃ ^– /1% CO₂ Ringer or 20 mM HCO ₃ ^– alone. Stimulation of I _sc induced by unilateral (luminal or serosal) HCO ₃ ^– /CO₂ was inhibited by luminal or serosal 4-acetamido-4-isothiocyanostilbene-2,2-disulphonic acid (SITS) (0,25 mM) or by serosal Na⁺ removal, whereas amiloride (1 mM), luminal or serosal, had no effect. Acetazolamide (0.1 mM, both sides) inhibited stimulation of I _sc induced by luminal addition of HCO ₃ ^– /CO₂, whereas it was without effect when HCO ₃ ^– /CO₂ was added serosally or bilaterally. We reached the following conclusions, (a) Cl^– absorption is stimulated by HCO ₃ ^– /CO₂ buffer via an increase in intracellular HCO ₃ ^– concentration and/or pH_i changes consequent to the HCO ₃ ^– uptake mediated by HCO ₃ ^– transport systems operating on both cell membranes, (b) A Na⁺-dependent SITS-inhibitable HCO ₃ ^– transport mechanism operates at the basolateral membrane, (c) The transfer of HCO ₃ ^– through the luminal membrane is mediated by the carbonic anhydrase enzyme located on the brush-border membranes of the enterocyte: the movement of HCO ₃ ^– , via a SITS-sensitive transport system, occurs most likely in form of OH^–, which originates from the dehydration reaction of HCO ₃ ^– catalysed by the carbonic anhydrase. (d) There is no apparent amiloride-sensitive Na⁺/H⁺ antiporter on either cell membrane.This work was supported by a research grant from Ministero dell'Università e della Ricerca Scientifica e Tecnologica — Progetto di interesse nazionale e di rilevante interesse per lo sviluppo della Scienza     

Acetazolamide inhibition of basolateral base exit in rabbit renal proximal tubule S2 segment

The influence of the carbonic anhydrase inhibitor acetazolamide (ACZ) was investigated on HCO ₃ ^– transport mechanisms in the basolateral cell membrane of rabbit renal proximal tubule. Experiments were performed on isolated S2 segments using double-barrelled microelectrodes to measure cell membrane potential (V _b) and cell pH (pH_i) during step changes in bath perfusate ion concentrations. Peritubular application of ACZ (1 mmol/l) reduced the initial V _b response to 101 reduction of bath HCO ₃ ^– concentration only slightly, from +53.8±4.2 mV to+49.1±0.3 mV (n=5), but caused an intermittent overshooting repolarization in the secondary V _b response. In conjunction with these effects it left the initial pH_i response virtually unchanged but induced a secondary slow acidification. These observation indicate that — under the present experimental conditions — ACZ does not block the Na⁺-HCO ₃ ^– cotransporter but acts via inhibition of cytosolic carbonic anhydrase. This was confirmed by studying the effect of elevated intracellular HCO ₃ ^– concentrations under reduced flux conditions and by comparing the concentration dependence of the V _b response with the inhibition kinetics of cytosolic carbonic anhydrase. In contrast, peritubular ACZ inhibited Na⁺-independent Cl^–/HCO ₃ ^– exchange in the basolateral cell membrane of S2 segments directly in a similar way to that described in the preceding publication for S3 segments.     

The key role of the mitochondria-rich cell in Na+ and H+ transport across the frog skin epithelium

We have investigated the possibility that the mitochondria-rich (MR) cells participate in sodium and proton transport, when the frog skin epithelium is bathed on its apical side with solutions of low Na⁺ concentration, by comparing transport rates with morphological observations (MR cell number and MR cell pit surface area). Frogs were adapted to various salinities or the isolated skins were treated with the following hormones, deoxycorticosterone acetate (DOCA), arginine vasotocin (AVT) and oxytocin in order to modify the transport of sodium and hydrogen ions. Adaptation of the frogs (either 3–4 days or 7–10 days) to distilled water, NaCl (50 mmol/l), KCl (50 mmol/l) or Na₂SO₄ (25 mmol/l) solutions modified the Na⁺ transport rate and the morphology of the epithelium. The highest Na⁺ transport rates were found for the animals adapted to the Na⁺ free solutions and were correlated with an increase in the total MR cell pit-surface area (number of MR cells x individual cell pit-surface area). The KCl adaptated group showed the largest increase in sodium and proton transport and also presented a metabolic acidosis as reflected by plasma acidification (pCO₂ increase and HCO ₃ ^– decrease). Proton secretion and sodium absorption were also found to be stimulated by either serosal DOCA addition (10^–6 M) or during acidification of the epithelium by serosally applied CO₂. Na⁺ transport was enhanced by AVT (10^–6 M) or oxytocin (100mU/ml) when the skin was bathed on its apical side with a high Na⁺ containing solution (115 mmol/l), whereas these hormones did not exert any effect on Na⁺ transport when the apical solution was low in Na⁺ (0.5mmol/l). It is concluded that MR cells play a key role in Na⁺ and H⁺ transport through the frog skin epithelium when bathed on its apical side with a low Na⁺ containing solution. Distinct pathways for sodium transport through two cell types (MR cells and granular cells) are proposed depending on the Na⁺ concentration of the solution bathing the apical side of the epithelium.