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1.
Amiloride in the mucosal fiuid (at concentrations of 5 · 10?6 M to 10?4 M) reversibly stimulates the HCO3?-dependent moiety of the short-circuiting current (Isc) in ouabain-treated turtle bladders bathed by Na-free Ringer solutions with or without Cl?.This effect is uniquely different from the known inhibitory effect of this agent on Na+ transport. Thus, any comprehensive hypothesis on the action of amiloride over a wide dosage-response fange should take into account its effect on HCO3? transport. 相似文献
2.
Hitoshi Harada Yoshihito Kanai Masashi Anzai Yasunobu Suketa 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1991,1092(3):404-407
The role of cAMP in regulation of intracellular pH in the confluent LLC-PK1 cells was investigated. DibutyrylcAMP and forskolin induce intracellular acidification. This acidification is inhibited by DIDS and ethacrynic acid, inhibitors of Na+-independent Cl?/HCO3? exchange, and by removal of extracellular Cl?. In addition, Bt2 cAMP causes Cl? entry into LLC-PK1 cells. These results suggest that cAMP activates Cl? transport, namely Na+-independent Cl?/HCO3? exchange, which participates in pHi regulation. 相似文献
3.
The effect of changing [K+], [Na+] and [Cl?] in nutrient solution was studied in bullfrog antrum with and without HCO3? in nutrient. In 25 mM HCO3? (95% O2/5% CO2) and in zero HCO3? (100% O2), nutrient pH was maintained at 7.3. Changing from 4 to 40 mM K+ or from 81 to 8.1 mM Cl? gave a decrease 10 min later in transmucosal PD (nutrient became more negative) — a normal response. These responses were less in zero than in 25 mM HCO3?. A decrease from 102 to 8 mM Na+ decreased PD (anomalous response of electrogenic NaCl symport). This effect was attenuated or eliminated in zero HCO3?. In contrast, change from 4 to 40 mM K+ gave initial anomalous PD response and change from 102 to 8 mM Na+, initial normal PD response with either zero or 25 mM HCO3?. Both responses were associated with (Na+ + K+)-ATPase pump and were greater in zero than in 25 mM HCO3?. Initial PD increases in zero HCO3? are explained as due to increase in the resistance of passive conductance and/or NaCl symport pathways. Thus, removal of HCO3? modifies conductance pathways of nutrient membrane. 相似文献
4.
Gerhard Ehrenspeck 《生物化学与生物物理学报:生物膜》1982,684(2):219-227
Ouabain-treated turtle bladders bathed on both surfaces by identical HCO3?/CO2-containing, Cl?-free Na+ media exhibit a short-circuit current (Isc) and transepithelial potential (p.d.) serosa electronegative to mucosa. Addition of 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cyclic nucleotide phosphodiesterase, rapidly reverses the direction of the Isc and p.d.. The IBMX-induced reversal of Isc and p.d. is (1) dependent on the presence of HCO3? (and CO2) in the serosal bathing fluid, (2) independent of Na+ and other ions in the bathing medium, (3) decreased by inhibitors of carbonic anhydrase or oxidative metabolism, (4) increased by the serosal addition of cyclic AMP or the disulfonic stilbene, SITS. The results constitute evidence that the reversed Isc elicited by IBMX represents electrogenic secretion of HCO3?. 相似文献
5.
C C Lambert 《Developmental biology》1982,91(2):257-262
Ascidia callosa sperm are triggered to undergo initiation of the sperm reaction (mitochondrial swelling) by increasing the pH or lowering the Na+ concentration of the medium. The optimal [Na+] for acid release is 20 mM with excellent correlation between acid release and initiation of morphological changes. Increasing the [K+] to around 20 mM inhibits acid release when applied up to 1 min after triggering the sperm but with less inhibition at 2 and 4 min, suggesting that K+ inhibits initiation of acid release rather than acid release itself. Acid release and the sperm reaction can also be triggered by Cl?-free (NO?3 or glutamate substituted) seawater (SW). Cl? efflux accompanies H+ efflux with twice as many Cl? being released as H+. Both H+ and Cl? release in Cl?-free SW are dependent upon CO2 being present in HCO?3-free medium, suggesting that H+ efflux is in part Cl? and HCO?3-mediated. However, the chloride channel blocking agent SITS has no effect on H+ release and augments Cl? release. Acid release results in a substantial increase in internal pH as determined by partitioning of 9-amino acridine. We envision acid release from ascidian sperm as involving two systems, the Na+-dependent acidification system of unreacted sperm and the Cl?- and HCO?3-mediated H+ release at activation. The mechanism controlling acid release would then involve inactivation of the internal acidification process and activation of the chloride-bicarbonate-mediated alkalinization process. 相似文献
6.
Effect of changing [K+], [Na+] and [Cl?] in nutrient solution on potential difference (PD) and resistance was studied in bullfrog antrum with and without nutrient HCO3? but with 95% O2/5% CO2 in both cases. In both cases, changing from 4 to 40 mM K+ gave about the same initial PD maximum (anomalous response) which was followed by a decrease below control level. Latter effect was much less with zero than with 25 mM HCO3?. Changing from 102 to 8 mM Na+ gave initial normal PD response about the same in both cases. However, 10 min later the change in PD with zero HCO3? was insignificant but with 25 mM HCO3? the PD decreased (anomalous response of electrogenic NaCl symport). PD maxima due to K+ and Na+ were largely related to ( pump. Changes in nutrient Cl? from 81 to 8.1 mM gave only a decrease in PD (normal response). Initial PD increases are explained by relative increases in resistance of simple conductance pathways and of parallel pathways of ( pump and Na+/Cl? symport. Removal of HCO3? and concurrent reduction of pH modify resistance of these pathways. 相似文献
7.
H+ extrusion by the isolated skins of two amphibia, Rana ridibunda and Bufo bufo was studied in order to test for the presence of exchange mechanisms of the type Na+/H+ and Cl?/HCO3?, which have been described in several epithelial structures. The preparations were mounted in chambers of the Ussing type, so that the short-circuit current could be used as a function of Na+ transport and the pH-stat technique was utilized to determine the rates of H+ extrusion under different experimental conditions. These conditions were either the withdrawal of the ions intervening in the mentioned exchanges (Cl- or Na+, or the addition of drugs with well-known effects on Na+ uptake and transport (antidiuretic hormone and amiloride).In the frog skin, H+ excretion was detected in solutions containing either Cl? or SO42?, with identical rates. Again, Na+ substitution by Mg2+ had no effect on H+ excretion rates, neither did the suppression of Na+ influx by amiloride or its stimulation by antidiuretic hormone. These experiments were repeated with similar results in gland-free preparations of the epidermis of frog skin separated from the corion by the action of collagenase.Experiments in toad skin showed that H+ excretion could not be detected when Cl? was present in the outer medium, but became apparent if an impermeant anion, SO42?, was used. This observation is compatible with the existence of an exchange mechanism of the type Cl?/HCO3?. Secondly, in these preparations H+ extrusion increased after stimulation with antidiuretic hormone and decreased when amiloride was used or when Na+ was substituted by Mg2+, suggesting that at least a fraction of the total H+ efflux is linked to Na+ influx. In the isolated frog skin this mechanism does not seem to be operative. 相似文献
8.
Poliovirus-induced intracellular alkalinization involves a proton ATPase and protein phosphorylation
We reported previously that poliovirus infection induces alkalinization in HeLa cells and that an alkaline intracellular pH (pHi) promoted viral replication. Additional experiments were carried out to understand the underlying mechanism. Virus-infected or control monolayer cultures were incubated with nominally bicarbonate-free Eagle's minimal essential medium (MEM) buffered with N-2-hydroxyethylpiperazine-N-3-ethanesulfonic acid (HEPES), and immediately following preincubations, changes in pHi were monitored via benzoic acid uptake around 2 h postinfection. The absence of pH increase in cells infected with ultraviolet light-inactivated virus (UV-virus) indicated that viral gene expression was required for this effect. On the other hand, lack of effect of 3 mM guanidine, an inhibitor of poliovirus-specific RNA but not protein synthesis, suggested that translation of input viral genome RNA is sufficient for the pH increase. Activation of Na+/H+ exchange, Cl?HCO?3 exchange, or H+-ATPase was considered as possible mechanisms by which alkalinization occurs in virus-infected cells. Na+/H+ exchange was excluded because the pH effect occurred in a Na+/H+ exchange deficient HeLa cell mutant. Similarly, Cl?/HCO?3 exchange was excluded because virus-specific alkalinization was evident in the presence of Cl? or bicarbonate deficient medium and was not associated with an increase in HCO?3 uptake or a decrease in Cl? uptake. Lack of dependence on Na+, abrogation by 10 μM 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl), and resistance to 1 mM vandate suggested that this effect was due to the activation of a vacuolar-type (V) proton ATPase. Studies using protein kinase inhibitors indicated that activation of the ATPase in virus-infected cells probably involved protein kinase C-mediated phosphorylation. © 1993 Wiley-Liss, Inc. 相似文献
9.
Abstract: The role of transmembrane processes that are dependent on external anions in the regulation of cerebral intracellular pH (pHi), high-energy metabolites, and lactate was investigated using 31P and 1H NMR spectroscopy in an ex vivo brain slice preparation. During oxygenated superfusion, removal of external HCO3?/CO2 in the presence of Na+ led to a sustained split of the inorganic phosphate (Pi) peak so that the pHi indicated by one part of the peak was 0.38 pH units more alkaline and by the other part 0.10 pH units more acidic at 5 min than in the presence of HCO3?. The pH in the compartment with a higher pHi value returned to 7.29 ± 0.04 by 10.5 min of superfusion in a HCO3?-free medium, whereas the pHi in an acidic compartment was reduced to 7.02. In the presence of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid or the absence of external Cl?, removal of HCO3? caused alkalinization without split of the Pi peak. Both treatments reduced the rate of pHi normalization following alkalinization. Simultaneous omission of external HCO3? and Na+ did not inhibit alkalinization of the pHi following CO2 exit. All these data show that the acid loading mechanism at neutral pHi is mediated by an Na+-independent anion transport. During severe hypoxia, pHi dropped from 7.29 ± 0.05 to 6.13 ± 0.16 and from 7.33 ± 0.03 to 6.67 ± 0.05 in the absence and presence of HCO3?, respectively, in Na+-containing medium. Lactate accumulated to 18.7 ± 2.8 and 19.6 ± 1.5 mmol/kg under the respective conditions. In the HCO3?-free medium supplemented with 1 mM amiloride, the pHi fell only to 6.94 ± 0.08 despite the lactate concentration of 18.9 ± 2.4 mmol/kg. Acidification caused by hypoxia was also small in the slice preparations superfused in the absence of both HCO3? and Cl?, as the pHi was 7.01 ± 0.12 at a lactate concentration of 24.5 ± 2.4 mmol/kg. These data indicate that apart from anaerobic glucose metabolism, separate acidifying mechanisms are functioning during hypoxia under these conditions. Recovery of phosphocreatine levels following reoxygenation was >75% relative to the prehypoxic level in the slice preparations superfused in the absence of HCO3? but <47% in those preparations superfused without HCO3? and Cl?. This indicates that either neutral pHi or absence of Cl? during hypoxia was deleterious to the energy metabolism. The present data indicate that Cl?/HCO3? exchange mechanisms have distinct roles in cerebral H+ homeostasis depending on the level of pHi and energy state. 相似文献
10.
R. J. RITCHIE 《Plant, cell & environment》1992,15(2):179-184
The kinetics of the light-driven Cl? uptake pump of Synechococcus R-2 (PCC 7942) were investigated. The kinetics of Cl? uptake were measured in BG-11 medium (pHo, 7·5; [K+]o, 0·35 mol m?3; [Na+]o, 18 mol m?3; [Cl?]o, 0·508 mol m?3) or modified media based on the above. Net36Cl? fluxes (?Cl?o,i) followed Michaelis-Menten kinetics and were stimulated by Na+ [18 mol m?3 Na+ BG-11 ?Cl?max= 3·29±0·60 (49) nmol m?2 s?1 versus Na+-free BG-11 ?Cl?max= 1·02±0·13 (54) nmol m?2 s?1] but the Km was not significantly different in the presence or absence of Na+ at pHo 10; the Km was lower, but not affected by the presence or absence of Na+ [Km = 22·3±3·54 (20) mmol m?3]. Na+ is a non-competitive activator of net ?Cl?o,i. High [K+]o (18 mol m?3) did not stimulate net ?Cl?o,i or change the Km in Na+-free medium. High [K+]o (18 mol m?3) added to Na+ BG-11 medium decreased net ?Cl?o,i [18 mol m?3K+ BG-11; ?Cl?max= 2·50±0·32 (20) nmol m?2 s?1 versus BG-11 medium; ?Cl?max= 3·35±0·56 (20) nmol m?2 s?1] but did not affect the Km 55·8±8·100 (40) mmol m?3]. Na+-stimulation of net ?Cl?o,i followed Michaelis-Menten kinetics up to 2–5 mol m?3 [Na+]o but higher concentrations were inhibitory. The Km for Na+-stimulation of net ?Cl?o,i [K1/2(Na+)] was different at 47 mmol m?3 [Cl?]o (K1/2[Na+] = 123±27 (37) mmol m?3]. Li+ was only about one-third as effective as Na+ in stimulating Cl? uptake but the activation constant was similar [K1/2(Li+) = 88±46 (16) mmol m?3]. Br? was a competitive inhibitor of Cl? uptake. The inhibition constant (Ki) was not significantly different in the presence and absence of Na+. The overall Ki was 297±23 (45) mmol m?3. The discrimination ratio of Cl? over Br? (δCl?/δBr?) was 6·38±0·92 (df = 147). Synechococcus has a single Na+-stimulated Cl? pump because the Km of the Cl? transporter and its discrimination between Cl? and Br? are not significantly different in the presence and absence of Na+. The Cl? pump is probably driven by ATP. 相似文献
11.
H. Onken K. Graszynski A. Johannsen M. Putzenlechner S. Riestenpatt C. Schirmer D. Siebers W. Zeiske 《Helgoland Marine Research》1995,49(1-4):715-725
In the present article we review our findings on split lamella preparations of crab gills mounted in modified Ussing-chambers with respect to mechanistic and ecophysiological aspects. The leaky gill epithelium of shore crabs adapted to brackish water absorbs Na+ and Cl? in a coupled mode, and shows similarities to other salt-absorbing epithelia exposed to moderately diluted media. The results so far obtained for NaCl uptake across the gills of the shore crab are compatible with a transport model where two cell types operate in parallel, one displaying cotransport-like NaCl absorption, similar to that in the thick ascending limb of Henle's loop of the mammalian mephron, and the other one with characteristics of amiloride-sensitive, channel-mediated Na+ uptake by frog skin. Although there is no clear evidence for the apical mechanisms in this model, it may serve as a good basis for more detailed studies in the future. The moderately tight gill epithelium of freshwater adapted Chinese crabs absorbs Na+ and Cl? independently from each other, and shows similarities to other salt-absorbing epithelia exposed to freshwater. The characteristics of a positive, Na+-dependent short-circuit current with externally Cl?-free saline indicate that active Na+ uptake proceeds in a frog-skin-like mode via apical Na+-channels and the basolateral Na+/K+-pump. The nature of a negative short-circuit current with external Cl?-saline indicates that active and Na+-independent Cl? uptake is driven by an apical V-type H+-pump and proceeds via apical Cl?/ HCO3 ?-exchange and basolateral Cl?-channels. 相似文献
12.
Rattana Chaimana Jarinthorn Teerapornpuntakit Walailak Jantarajit Kornkamon Lertsuwan Saowalak Krungchanuchat Nattapon Panupinthu Nateetip Krishnamra Narattaphol Charoenphandhu 《Biochemistry and Biophysics Reports》2021
Parathyroid hormone (PTH) has previously been shown to enhance the transepithelial secretion of Cl? and HCO3? across the intestinal epithelia including Caco-2 monolayer, but the underlying cellular mechanisms are not completely understood. Herein, we identified the major signaling pathways that possibly mediated the PTH action to its known target anion channel, i.e., cystic fibrosis transmembrane conductance regulator anion channel (CFTR). Specifically, PTH was able to induce phosphorylation of protein kinase A and phosphoinositide 3-kinase. Since the apical HCO3? efflux through CFTR often required the intracellular H+/HCO3? production and/or the Na+-dependent basolateral HCO3? uptake, the intracellular pH (pHi) balance might be disturbed, especially as a consequence of increased endogenous H+ and HCO3? production. However, measurement of pHi by a pH-sensitive dye suggested that the PTH-exposed Caco-2 cells were able to maintain normal pH despite robust HCO3? transport. In addition, although the plasma membrane Na+/K+-ATPase (NKA) is normally essential for basolateral HCO3? uptake and other transporters (e.g., NHE1), PTH did not induce insertion of new NKA molecules into the basolateral membrane as determined by membrane protein biotinylation technique. Thus, together with our previous data, we concluded that the PTH action on Caco-2 cells is dependent on PKA and PI3K with no detectable change in pHi or NKA abundance on cell membrane. 相似文献
13.
Katsuhisa Ikeda Motoaki Ishigaki Dazheng Wu Hiroshi Sunose Tomonori Takasaka 《Journal of cellular physiology》1995,163(1):204-209
In the dispersed acinar cells of the submucosal nasal gland in the guinea pig, intracellular Na+ concentration ([Na+]i) was measured with a microfluorimetric imaging method and the cytosolic indicator dye, sodium-binding benzofuran isophthalate, under HCO3?-free conditions. In the unstimulated condition, the [Na+]i was averaged to 12.8 ± 5.2 mM. Addition of 100 μM ouabain or removal of external K+ caused an increase in [Na+]i. Replacement of external Cl? with NO3? or addition of 0.5 mM furosemide reversibly decreased the [Na+]i. The recovery process from the reduced [Na+]i was inhibited by removal of either K+ or Cl? in the bath solution. These findings indicate the presence of a continuous influx of Na+ coupled with K+ and Cl? movement. Application of acetylcholine (ACh, 1 μM) caused an increase in [Na+]i by about 15–20 mM, which was completely inhibited by addition of 10 μM atropine. Increased cytosolic Na+ induced by ACh was extruded by the Na+-K+ pump. Removal of external Cl? and addition of 50 μM dimethylamiloride inhibited ACh-induced increase in [Na+]i by about 66% and 19%, respectively. In both unstimulated and stimulated state, Na+-K+ pump, Na-K-Cl cotransport, and Na+-H+ exchange play a critical role in maintaining intracellular electrolyte environment and in controlling a continuous secretion of nasal fluids. © 1995 Wiley-Liss, Inc. 相似文献
14.
Raquel L. Bernardino Ana D. Martins Sílvia Socorro Marco G. Alves Pedro F. Oliveira 《The Journal of membrane biology》2013,246(12):877-883
The formation of competent spermatozoa is a complex event that depends on the establishment of adequate environments throughout the male reproductive tract. This includes the control of bicarbonate (HCO3 ?) concentration, which plays an essential role in the maintenance of extracellular and intracellular pH (pHi) values. Diabetes mellitus alters pHi regulation in mammalian cells, mainly by altering the activity of ion transporters, particularly HCO3 ?-dependent mechanisms. Yet, little is known about the effects of this pathology and its prodromal stage, prediabetes, on the membrane transport mechanisms of male reproductive tract cells. Herein, we analyzed protein and mRNA levels of the most relevant HCO3 ? transporters of the SLC4 family [anion exchanger 2 (AE2), Na+-driven Cl?/HCO3 ? exchanger (NDCBE), electrogenic Na+/HCO3 ? cotransporter 1 (NBCe1), electroneutral Na+/HCO3 ? cotransporter 1 (NBCn1)] in the testis and epididymis of a prediabetic animal model. Firstly, we identified the HCO3 ? transporters of the SLC4 family, in both testicular and epididymal tissue. Secondly, although no alterations were detected in protein expression, mRNA levels of NBCe1, NBCn1 and NDCBE were significantly increased in the testis of prediabetic rats. On the other hand, in the epididymis, prediabetes caused an increase of AE2 and a decrease of NDCBE protein levels. These alterations may be translated into changes of HCO3 ? transepithelial epididymal fluxes in vivo, which may represent a threat for sperm survival. Moreover, these results provide evidence of the molecular mechanism that may be responsible for the significant increase in abnormal sperm morphology already reported in prediabetic rats. 相似文献
15.
The monovalent ion transport systems of an immortalized insect cell line (CHE) have been investigated. These cells are unusual in that unlike most vertebrate cells, their normal extracellular environment consists of high potassium and low sodium concentrations. CHE cells maintained high intracellular [K+] through both a furosemide-inhibitable and a vanadate-inhibitable transport system. Intracellular exchangeable [Na+] was slightly lower than the extracellular [Na+] and was maintained at this level through a vanadate-sensitive transport system. Na+ uptake was also inhibited by furosemide: however, the stoichiometry of furosemide-sensitive Na+ uptake when compared with furosemide-sensitive K+ uptake indicated that these cations are not cotransported. 4,4′-Diisothiocyano-2,2′-disulfonic acid stilbene (DIDS) inhibited Na+, K+, and Cl? uptake. Vanadate and furosemide decreased cytoplasmimic pH, while cytoplasmic pH increased in the presence of DIDS. A model is presented explaining how Na+, K+, Cl?, H+ and HCO3 ? fluxes are regulated in these cells. 相似文献
16.
17.
Cell pH regulation was investigated in the T84 cell line derived from epithelial colon cancer. Cell pH was measured by ratiometric
fluorescence microscopy using the fluorescent probe BCECF. Basal pH was 7.17 ± 0.023 (n= 48) in HEPES Ringer. After acidification by an ammonium pulse, cell pH recovered toward normal at a rate of 0.13 ± 0.011
pH units/min in the presence of Na+, but in the absence of this ion or after treatment with 0.1 mm hexamethylene amiloride (HMA) no significant recovery was observed, indicating absence of Na+ independent H+ transport mechanisms in HEPES Ringer. In CO2/HCO−
3 Ringer, basal cell pH was 7.21 ± 0.020 (n= 35). Changing to HEPES Ringer, a marked alkalinization was observed due to loss of CO2, followed by return to the initial pH at a rate of −0.14 ± 0.012 (n= 8) pH/min; this return was retarded or abolished in the absence of Cl− or after addition of 0.2 mm DIDS, suggesting extrusion of bicarbonate by Cl−/HCO−
3 exchange. This exchange was not Na+ dependent. When Na+ was added to cells incubated in 0 Na+ Ringer while blocking Na+/H+ exchange by HMA, cell alkalinization by 0.19 ± 0.04 (n= 11) pH units was observed, suggesting the presence of Na+/HCO−
3 cotransport carrying HCO−
3 into these cells, which was abolished by DIDS. These experiments, thus, show that Na+/H+ and Cl−/HCO−
3 exchange and Na+/HCO−
3 cotransport participate in cell pH regulation in T84 cells.
Received: 3 April 2000/Revised: 22 June 2000 相似文献
18.
The disulfonic stilbene (4-acetamido-4′-isothiocyano-2,2′-disulfonic stilbene) is found to be more potent than acetazolamide as an anion transport inhibitor in the turtle bladder, but less potent than acetazolamide as a carbonic anhydrase inhibitor. The anion-dependent (HCO3-−, Cl−) moeity of the short-circuiting current is eliminated by 4-acetamido-4′-isothiocyano-2,2′-disulfonic stibene, but only after its addition to the serosal bathing fluid. Whereas 4-acetmido-4′-isothiocyano-2,2′-disulfonic stilbene has no effect om Na+transport across the bladder, it is more potent than ouabain as an inhibitor of microsomal (Na++K+)-ATPase of both turtle bladder and eel electric organ. 相似文献
19.
Mark O. Bevensee Michael Apkon Walter F. Boron 《The Journal of general physiology》1997,110(4):467-483
In the preceding paper (Bevensee, M.O., R.A. Weed, and W.F. Boron. 1997. J. Gen. Physiol. 110: 453–465.), we showed that a Na+-driven influx of HCO3
− causes the increase in intracellular pH (pHi) observed when astrocytes cultured from rat hippocampus are exposed to 5% CO2/17 mM HCO3
−. In the present study, we used the pH-sensitive fluorescent indicator 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and the perforated patch-clamp technique to determine whether this transporter is a Na+-driven Cl-HCO3 exchanger, an electrogenic Na/HCO3 cotransporter, or an electroneutral Na/HCO3 cotransporter. To determine if the transporter is a Na+-driven Cl-HCO3 exchanger, we depleted the cells of intracellular Cl− by incubating them in a Cl−-free solution for an average of ∼11 min. We verified the depletion with the Cl−-sensitive dye N-(6-methoxyquinolyl)acetoethyl ester (MQAE). In Cl−-depleted cells, the pHi still increases after one or more exposures to CO2/HCO3
−. Furthermore, the pHi decrease elicited by external Na+ removal does not require external Cl−. Therefore, the transporter cannot be a Na+-driven Cl-HCO3 exchanger. To determine if the transporter is an electrogenic Na/ HCO3 cotransporter, we measured pHi and plasma membrane voltage (Vm) while removing external Na+, in the presence/absence of CO2/HCO3
− and in the presence/absence of 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS). The CO2/HCO3
− solutions contained 20% CO2 and 68 mM HCO3
−, pH 7.3, to maximize the HCO3
− flux. In pHi experiments, removing external Na+ in the presence of CO2/HCO3
− elicited an equivalent HCO3
− efflux of 281 μM s−1. The HCO3
− influx elicited by returning external Na+ was inhibited 63% by DIDS, so that the predicted DIDS-sensitive Vm change was 3.3 mV. Indeed, we found that removing external Na+ elicited a DIDS-sensitive depolarization that was 2.6 mV larger in the presence than in the absence of CO2/ HCO3
−. Thus, the Na/HCO3 cotransporter is electrogenic. Because a cotransporter with a Na+:HCO3
− stoichiometry of 1:3 or higher would predict a net HCO3
− efflux, rather than the required influx, we conclude that rat hippocampal astrocytes have an electrogenic Na/HCO3 cotransporter with a stoichiometry of 1:2. 相似文献
20.
A. A. Bernardo F. T. Kear J. A. Stim Y. Y. Qiu O. S. Ruiz H. Weidman J. A. L. Arruda 《The Journal of membrane biology》1995,145(1):67-74
The activity of the Na-H antiporter is inhibited by cyclic AMP-dependent protein kinase A (cAMP.PKA). The inhibitory effect of PKA on the Na-H antiporter is mediated through a regulatory protein that can be dissociated from the antiporter by limited protein digestion. PKA also inhibits the activity of the Na+/ HCO 3 ? cotransporter. We investigated whether the activity of Na+/HCO 3 ? cotransporter and the effect of PKA on this transporter may also be regulated by limited protein digestion. In rabbit renal cortical basolateral membranes (BLM) and in solubilized BLM reconstituted in liposomes (proteoliposomes), trypsin (100 μg) increased 22Na uptake in the presence of HCO3 but not in the presence of gluconate, indicating that trypsin does not alter diffusive 22Na uptake but directly stimulates the Na+/HCO 3 ? cotransporter activity. In proteoliposomes phosphorylated with ATP, the catalytic subunit (CSU) of cAMP-PKA decreased the activity of the Na+/HCO 3 ? cotransporter (expressed as nanomoles/mg protein/3s) from 23 ± 10 to 14 ± 6 (P < 0.01). In the presence of trypsin, the inhibitory effect of CSU of cAMP-PKA on the activity of Na+/HCO 3 ? cotransporter was blunted. To identify a fraction that was responsible for the inhibitory effect of the CSU on the Na+/HCO 3 ? cotransporter activity, solubilized proteins were separated by size exclusion chromatography. The effect of CSU of cAMP-PKA on the Na+/HCO 3 ? cotransporter activity was assayed in proteoliposomes digested with trypsin with the addition of a fraction containing the 42 kDa protein (fraction S+) or without the 42 kDa protein (fraction S?). With the addition of fraction S?, the CSU of cAMP-PKA failed to inhibit the Na+/HCO 3 ? cotransporter activity (control 27 ± 6, CSU 27 ± 3) while the addition of fraction S+ restored the inhibitory effect of CSU (27 ± 6 to 3 ± 0.3 P < 0.01). The CSU of cAMP-PKA phosphorylated several proteins in solubilized protein including a 42 kDa protein. Fluorescein isothiocyanate (FITC) labels components of the Na+/HCO 3 ? cotransporter including the 56 kDa and 42 kDa proteins. In trypsin-treated solubilized protein the 42 kDa protein was not identified with FITC labeling. The results demonstrate that the activity of the Na+/HCO 3 ? cotransporter is regulated by protein(s) which mediates the inhibitory effect of PKA. Limited protein digestion can dissociate this protein from the cotransporter. 相似文献