scholarly journals Extracellular Cl− modulates shrinkage-induced activation of Na+/H+exchanger in rat mesangial cells

2000 ◽  
Vol 278 (6) ◽  
pp. C1218-C1229 ◽  
Author(s):  
Yukio Miyata ◽  
Shigeaki Muto ◽  
Satoru Yanagiba ◽  
Yasushi Asano
Keyword(s):  
Benzoic Acid ◽  
Intracellular Ph ◽  
Recovery Rate ◽  
Mesangial Cells ◽  
Ph Sensitive ◽  
Extrusion Rate ◽  
Wide Range ◽  
Dependent Cell ◽  
Acid Extrusion ◽  
Dependent Processes

To examine the effect of hyperosmolality on Na+/H+ exchanger (NHE) activity in mesangial cells (MCs), we used a pH-sensitive dye, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM, to measure intracellular pH (pHi) in a single MC from rat glomeruli. All the experiments were performed in CO2/[Formula: see text]-free HEPES solutions. Exposure of MCs to hyperosmotic HEPES solutions (500 mosmol/kgH2O) treated with mannitol caused cell alkalinization. The hyperosmolality-induced cell alkalinization was inhibited by 100 μM ethylisopropylamiloride, a specific NHE inhibitor, and was dependent on extracellular Na+. The hyperosmolality shifted the Na+-dependent acid extrusion rate vs. pHi by 0.15–0.3 pH units in the alkaline direction. Removal of extracellular Cl− by replacement with gluconate completely abolished the rate of cell alkalinization induced by hyperosmolality and inhibited the Na+-dependent acid extrusion rate, whereas, under isosmotic conditions, it caused no effect on Na+-dependent pHi recovery rate or Na+-dependent acid extrusion rate. The Cl−-dependent cell alkalinization rate under hyperosmotic conditions was partially inhibited by pretreatment with 5-nitro-2-(3-phenylpropylamino)benzoic acid, DIDS, and colchicine. We conclude: 1) in MCs, hyperosmolality activates NHE to cause cell alkalinization, 2) the acid extrusion rate via NHE is greater under hyperosmotic conditions than under isosmotic conditions at a wide range of pHi, 3) the NHE activation under hyperosmotic conditions, but not under isosmotic conditions, requires extracellular Cl−, and 4) the Cl−-dependent NHE activation under hyperosmotic conditions partly occurs via Cl− channel and microtubule-dependent processes.

scholarly journals Regulation of Intracellular pH in Single Rat Cortical Neurons in vitro: A Microspectrofluorometric Study

1993 ◽  
Vol 13 (5) ◽  
pp. 827-840 ◽  
Author(s):  
Yibing Ou-Yang ◽  
Pekka Mellergård ◽  
Bo K. Siesjö
Keyword(s):  
Steady State ◽  
Intracellular Ph ◽  
Cortical Neurons ◽  
Ph Sensitive ◽  
Disulfonic Acid ◽  
Extrusion Rate ◽  
Rat Cortical Neurons ◽  
Acid Extrusion

Intracellular pH (pHi) and the mechanisms of pHi regulation in cultured rat cortical neurons were studied with microspectrofluorometry and the pH-sensitive fluorophore 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein. Steady-state pHi was 7.00 ± 0.17 (mean ± SD) and 7.09 ± 0.14 in nominally HCO3− -free and HCO3−-containing solutions, respectively, and was dependent on extracellular Na+ and Cl−. Following an acid transient, induced by an NH1 prepulse or an increase in CO2 tension, pHi decreased and then rapidly returned to baseline, with an average net acid extrusion rate of 2.6 and 2.8 mmol/L/min, in nominally HCO3− -free and HCO3− -containing solutions, respectively. The recovery was completely blocked by removal of extracellular Na+ and was partially inhibited by amiloride or 5- N-methyl- N-isobutylamiloride. In most cells pHi recovery was completely blocked in the presence of harmaline. The recovery of pHi was not influenced by addition of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) or removal of Cl−. The rapid regulation of pHi seen following a transient alkalinization was not inhibited by amiloride or by removal of extracellular Na+, but was partially inhibited by DIDS and by removal of extracellular Cl−. The results are compatible with the presence of at least two different pHi-regulating mechanisms: an acid-extruding Na+/H+ antiporter, possibly consisting of different subtypes, and a passive Cl−/HCO3− exchanger, mediating loss of HCO3− from the cell.

scholarly journals Intracellular pH-regulating mechanism of the squid axon. Relation between the external Na+ and HCO-3 dependences.

1985 ◽  
Vol 85 (3) ◽  
pp. 325-345 ◽  
Author(s):  
W F Boron
Keyword(s):  
Intracellular Ph ◽  
Recovery Rate ◽  
Ion Pair ◽  
Squid Axon ◽  
Extrusion Rate ◽  
Pair Model ◽  
Buffering Power ◽  
Series Of Experiments ◽  
Acid Extrusion ◽  
External Ph

The intracellular pH-regulating mechanism of the squid axon was examined for its dependence on the concentrations of external Na+ and HCO3-, always at an external pH (pHo) of 8.0. Axons having an initial intracellular pH (pHi) of approximately 7.4 were internally dialyzed with a solution of pH 6.5 that contained 400 mM Cl- and no Na+. After pHi had fallen to approximately 6.6, dialysis was halted, thereby returning control of pHi to the axon. With external Na+ and HCO-3 present, intracellular pH (pHi) increased because of the activity of the pHi-regulating system. The acid extrusion rate (i.e., equivalent efflux of H+, JH) is the product of the pHi recovery rate, intracellular buffering power, and the volume-to-surface ratio. The [HCO3-]o dependence of JH was examined at three fixed levels of [Na+]o: 425, 212, and 106 mM. In all three cases, the apparent Jmax was approximately 19 pmol X cm-2 X s-1. However, the apparent Km (HCO3-) was approximately inversely proportional to [Na+]o, rising from 2.6 to 5.4 to 9.7 mM as [Na+]o was lowered from 425 to 212 to 106 mM, respectively. The [Na+]o dependence of JH was similarly examined at three fixed levels of [HCO3-]o: 12, 6, and 3 mM. The Jmax values did not vary significantly from those in the first series of experiments. The apparent Km (Na+), however, was approximately inversely related to [HCO3-]o, rising from 71 to 174 to 261 mM as [HCO3-]o was lowered from 12 to 6 to 3 mM, respectively. These results agree with the predictions of the ion-pair model of acid extrusion, which has external Na+ and CO3= combining to form the ion pair NaCO3-, which then exchanges for internal Cl-. When the JH data are replotted as a function of [NaCO3-]o, data from all six groups of experiments fall along the same Michaelis-Menten curve, with an apparent Km (NaCO3-) of 80 microM. The ordered and random binding of Na+ and CO3= cannot be ruled out as possible models, but are restricted in allowable combinations of rate constants.

scholarly journals Intermittent Hypoxia Inhibits Na+-H+ Exchange-Mediated Acid Extrusion Via Intracellular Na+ Accumulation in Cardiomyocytes

2018 ◽  
Vol 46 (3) ◽  
pp. 1252-1262 ◽  
Author(s):  
Huai-Ren Chang ◽  
Chih-Feng Lien ◽  
Jing-Ren Jeng ◽  
Jen-Che Hsieh ◽  
Chen-Wei Chang ◽  
...  
Keyword(s):  
Reperfusion Injury ◽  
Intracellular Calcium ◽  
Intracellular Ph ◽  
Recovery Rate ◽  
Intermittent Hypoxia ◽  
Ros Production ◽  
Tyrode Solution ◽  
Neonatal Cardiomyocytes ◽  
Cardioprotective Effects ◽  
Acid Extrusion

Background/Aims: Intermittent hypoxia (IH) has been shown to exert preconditioning-like cardioprotective effects. It also has been reported that IH preserves intracellular pH (pHi) during ischemia and protects cardiomyocytes against ischemic reperfusion injury. However, the exact mechanism is still unclear. Methods: In this study, we used proton indicator BCECF-AM to analyze the rate of pHi recovery from acidosis in the IH model of rat neonatal cardiomyocytes. Neonatal cardiomyocytes were first treated with repetitive hypoxia-normoxia cycles for 1-4 days. Cells were then acid loaded with NH4Cl, and the rate of pHi recovery from acidosis was measured. Results: We found that the pHi recovery rate from acidosis was much slower in the IH group than in the room air (RA) group. When we treated cardiomyocytes with Na+-H+ exchange (NHE) inhibitors (Amiloride and HOE642) or Na+-free Tyrode solution during the recovery, there was no difference between RA and IH groups. We also found intracellular Na+ concentration ([Na+]i) significantly increased after IH exposure for 4 days. However, the phenomenon could be abolished by pretreatment with ROS inhibitors (SOD and phenanathroline), intracellular calcium chelator or Na+-Ca2+ exchange (NCX) inhibitor. Furthermore, the pHi recovery rate from acidosis became faster in the IH group than in the RA group when inhibition of NCX activity. Conclusions: These results suggest that IH would induce the elevation of ROS production. ROS then activates Ca2+-efflux mode of NCX and results in intracellular Na+ accumulation. The rise of [Na+]i further inhibits the activity of NHE-mediated acid extrusion and retards the rate of pHi recovery from acidosis during IH.

Effects of angiotensin II and vasopressin on intracellular pH of glomerular mesangial cells

1988 ◽  
Vol 254 (6) ◽  
pp. F787-F794 ◽  
Author(s):  
M. B. Ganz ◽  
G. Boyarsky ◽  
W. F. Boron ◽  
R. B. Sterzel
Keyword(s):  
Angiotensin Ii ◽  
Intracellular Ph ◽  
Mesangial Cells ◽  
Ang Ii ◽  
Glomerular Mesangial Cells ◽  
Initial Value ◽  
Ph Change ◽  
Acid Load ◽  
Ethanesulfonic Acid ◽  
Acid Extrusion

We investigated changes in intracellular pH (pHi) of cultured rat glomerular mesangial cells (MCs) exposed to angiotensin II (ANG II) and arginine vasopressin (AVP). pHi of quiescent MCs, passage 2–5, and grown on glass cover slips, was assessed by spectrofluorometry using the pH-sensitive dye, 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). The steady-state pHi of MCs in a pH 7.4, HCO3-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution was 7.10 +/- 0.02 (n = 68) and in a pH 7.4, HCO3-containing solution, was 7.23 +/- 0.03 (n = 47) (P less than 0.01). The pHi recovery following an NH+4-induced acid load was inhibited by removal of Na+ from the bath or by addition of the amiloride analogue, ethyl isopropyl amiloride (EIPA). These effects were observed in MCs bathed in HEPES- or in HCO3-buffered solutions, consistent with the action of a Na+-H+ exchanger. When cells were bathed in HEPES, a 10-min exposure to ANG II or AVP (10(-10) to 10(-6) M) caused early and transient acidification of MCs (maximal pH change was -0.10), followed by gradual alkalinization (maximal pHi change +0.15 above the initial value). The increase of pHi was dependent on the presence of Na+ in the bath and was inhibited by EIPA. In the presence of HCO3, ANG II or AVP induced merely a small gradual acidification of MCs (pHi change -0.05). These findings demonstrate that MCs utilize a Na+-H+ exchanger for acid extrusion.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Effect of eccentric contraction-induced injury on force and intracellular pH in rat skeletal muscles

2002 ◽  
Vol 92 (1) ◽  
pp. 93-99 ◽  
Author(s):  
E. W. Yeung ◽  
J.-P. Bourreau ◽  
D. G. Allen ◽  
H. J. Ballard
Keyword(s):  
Intracellular Ph ◽  
Eccentric Contraction ◽  
Eccentric Contractions ◽  
Optimal Length ◽  
Extrusion Rate ◽  
Reduction In Force ◽  
Buffering Power ◽  
Acid Load ◽  
Rat Soleus Muscle ◽  
Acid Extrusion

The effect of eccentric contraction on force generation and intracellular pH (pHi) regulation was investigated in rat soleus muscle. Eccentric muscle damage was induced by stretching muscle bundles by 30% of the optimal length for a series of 10 tetani. After eccentric contractions, there was reduction in force at all stimulation frequencies and a greater reduction in relative force at low-stimulus frequencies. There was also a shift of optimal length to longer lengths. pHi was measured with a pH-sensitive probe, 2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein AM. pHi regulation was studied by inducing an acute acid load with the removal of 20–40 mM ammonium chloride, and the rate of pHi recovery was monitored. The acid extrusion rate was obtained by multiplying the rate of pHi recovery by the buffering power. The resting pHi after eccentric contractions was more acidic, and the rate of recovery from acid load post-eccentric contractions was slower than that from postisometric controls. This is further supported by the slower acid extrusion rate. Amiloride slowed the recovery from an acid load in control experiments. Because the Na+/H+ exchanger is the dominant mechanism for the recovery of pHi, this suggests that the impairment in the ability of the muscle to regulate pHiafter eccentric contractions is caused by decreased activity of the Na+/H+ exchanger.

scholarly journals Intracellular pH-regulating mechanism of the squid axon. Interaction between DNDS and extracellular Na+ and HCO3-.

1989 ◽  
Vol 93 (1) ◽  
pp. 123-150 ◽  
Author(s):  
W F Boron ◽  
R C Knakal
Keyword(s):  
Intracellular Ph ◽  
Kinetic Models ◽  
Ion Pair ◽  
Squid Axon ◽  
Maximal Inhibition ◽  
Extrusion Rate ◽  
Stilbene Derivative ◽  
Pair Model ◽  
Acid Extrusion ◽  
Best Fit

Intracellular pH (pHi) of the squid axon is regulated by a stilbenesensitive transporter that couples the influx of Na+ and HCO3- (or the equivalent) to the efflux of Cl-. According to one model, the extracellular ion pair NaCO3- exchanges for intracellular Cl-. In the present study, the ion-pair model was tested by examining the interaction of the reversible stilbene derivative 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) with extracellular Na+ and HCO3-. Axons (initial pHi approximately 7.4) were internally dialyzed with a pH 6.5 solution containing 400 mM Cl- but no Na+. After pHi, as measured with a glass microelectrode, had fallen to approximately 6.6, dialysis was halted. In the presence of both external Na+ and HCO3- (pHo = 8.0, 22 degrees C), pHi increased due to the pHi-regulating mechanism. At a fixed [Na+]o of 425 mM and [HCO3-]o of 12 mM, DNDS reversibly reduced the equivalent acid-extrusion rate (JH) calculated from the rate of pHi recovery. The best-fit value for maximal inhibition was 104%, and for the [DNDS]o at half-maximal inhibition, 0.3 mM. At a [Na+]o of 425 mM, the [HCO3-]o dependence of JH was examined at 0, 0.1, and 0.25 mM DNDS. Although Jmax was always approximately 20 pmol cm-2 s-1, Km(HCO3-) was 2.6, 5.7, and 12.7 mM, respectively. Thus, DNDS is competitive with HCO3-. At a [HCO3-]o of 12 mM, the [Na+]o dependence of JH was examined at 0 and 0.1 mM DNDS. Although Jmax was approximately 20 pmol cm-2 s-1 in both cases, Km(Na+) was 71 and 179 mM, respectively. At a [HCO3-]o of 48 mM, Jmax was approximately 20 pmol cm-2 s-1 at [DNDS]o levels of 0, 0.1, and 0.25 mM. However, Km(Na+) was 22, 45, and 90 mM, respectively. Thus, DNDS (an anion) is also competitive with Na+. The results are consistent with simple competition between DNDS and NaCO3-, and place severe restrictions on other kinetic models.

Depolarization-induced alkalinization in proximal tubules. I. Characteristics and dependence on Na+

1989 ◽  
Vol 256 (2) ◽  
pp. F342-F353 ◽  
Author(s):  
A. W. Siebens ◽  
W. F. Boron
Keyword(s):  
Intracellular Ph ◽  
Initial Rate ◽  
Basolateral Membrane ◽  
Ph Sensitive ◽  
Proximal Tubules ◽  
Ambystoma Tigrinum ◽  
Tiger Salamander ◽  
Additive Effects ◽  
Basolateral Membranes ◽  
Dependent Processes

We used intracellular pH-sensitive and voltage microelectrodes to examine the effects of depolarization on intracellular pH (pHi) in isolated perfused proximal tubules from the tiger salamander Ambystoma tigrinum. Tubules were depolarized by raising [K+] in the bath (b) or lumen (l), or by adding Ba2+ (1 mM) to the bath or lumen, always in nominally HCO3-free solutions. Increasing [K+]b from 2.5 to 50 mM caused the basolateral membrane to depolarize by an average of 45 mV, and pHi to increase by 0.23 over 3 min. Similar alkalinization was observed when basolateral Ba2+ (1 mM) was used to depolarize the cell at constant extracellular [K+], suggesting that the alkalinization observed during exposure to elevated [K+]b results from depolarization rather than an increase in [K+]b. The initial rate of depolarization-induced alkalinization (DIA) was proportional to the magnitude of the depolarization, regardless of whether tubules were depolarized by elevated [K+]b, elevated [K+]l, or by basolateral Ba2+. An exception was the initial rate of the alkalinization caused by 1 mM luminal Ba2+, which was more than 10-fold greater than that predicted from the depolarization. The voltage and pHi responses to basolateral Ba2+ were smaller in some tubules than others, as were the responses to elevated [K+]l. Tubules with small responses to 1 mM [Ba2+]b had large responses to 50 mM [K+]l, whereas tubules with large responses to 1 mM [Ba2+]b had small responses to 50 mM [K+]l. This variability can be accounted for by differences in the luminal K+ conductance. The DIA was partially inhibited by removal of Na+ from only the lumen or only the bath, but completely inhibited by bilateral Na+ removal. We conclude that the depolarization-induced alkalinization results from additive effects of Na+-dependent processes at both the luminal and basolateral membranes.

Shrinkage-induced activation of Na(+)-H+ exchange in barnacle muscle fibers

1994 ◽  
Vol 266 (6) ◽  
pp. C1744-C1753 ◽  
Author(s):  
B. A. Davis ◽  
E. M. Hogan ◽  
W. F. Boron
Keyword(s):  
Intracellular Ph ◽  
Muscle Fibers ◽  
Single Muscle ◽  
Extrusion Rate ◽  
Barnacle Muscle ◽  
Hypertonic Solutions ◽  
Acid Extrusion ◽  
Ph Microelectrodes ◽  
Barnacle Muscle Fibers ◽  
Exchange Activity

We examined the effect of shrinkage on Na(+)-H+ exchange in single muscle fibers at intracellular pH (pHi) values of 6.8, 7.2, and 7.6 using pH microelectrodes and internal dialysis. Under normotonic conditions (975 mosmol/kgH2O) at pHi 6.8, the amiloride-sensitive acid-extrusion rate (JAmil/s) averaged 17 microM/min. Exposure to hypertonic solutions (1,600 mosmol/kgH2O) increased JAmil/s to 304 microM/min at pHi 6.8. At pHi approximately 7.2 and 7.6, hypertonicity increased JAmil/s from approximately 0 to approximately 172 microM/min (pHi 7.2) and approximately 0 to approximately 90 microM/min (pHi 7.6). Thus, under normotonic conditions, Na(+)-H+ exchange activity is slight at pHi approximately 6.8 and virtually nil at higher pHi values. Shrinkage stimulated Na(+)-H+ exchange, more at low pHi values. We also examined the Cl- dependence of the Na(+)-H+ exchanger's response to shrinkage. Our results indicate that shrinkage-induced activation of Na(+)-H+ exchange requires Cl-, specifically intracellular Cl-. These results establish that shrinkage is both pHi dependent and requires intracellular Cl-.

scholarly journals Stoichiometry and ion dependencies of the intracellular-pH-regulating mechanism in squid giant axons.

1983 ◽  
Vol 81 (3) ◽  
pp. 373-399 ◽  
Author(s):  
W F Boron ◽  
J M Russell
Keyword(s):  
Intracellular Ph ◽  
Transport System ◽  
Extrusion Rate ◽  
Giant Axons ◽  
Constant Ph ◽  
Absolute Dependence ◽  
Standard Set ◽  
Series Of Experiments ◽  
The Mean ◽  
Acid Extrusion

The ion transport system responsible for intracellular pH (pHi) regulation in squid giant axons was examined in experiments with pH-sensitive microelectrodes and isotopic fluxes of Na+ and Cl-. In one study, axons were acid-loaded and the rate of the subsequent pHi recovery was used to calculate the acid extrusion rate. There was an absolute dependence of acid extrusion on external Na+, external HCO-3 (at constant pH), and internal Cl-. Furthermore, the dependence of the acid extrusion rate on each of these three parameters was described by Michaelis-Menten kinetics. Acid extrusion was stimulated by an acid pHi, required internal ATP, and was blocked by external 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS). Under a standard set of conditions (i.e., [HCO-3]o = 12 mM, pHo = 8.00, [Na+]o = 425 mM, [Cl-]i = 150 mM, [ATP]i = 4 mM, pHi = 6.5, and 16 degrees C), the mean acid extrusion rate was 7.5 pmol X cm-2 X s-1. In a second study under the above standard conditions, the unidirectional Na+ efflux (measured with 22Na) mediated by the pHi-regulating system was found to be approximately 0, whereas the mean influx was about 3.4 pmol X cm-2 X s-1. This net influx required external HCO-3, internal Cl-, and acid pHi, internal ATP, and was blocked by SITS. In the final series of experiments under the above standard conditions, the unidirectional Cl- influx (measured with 36Cl) mediated by the pHi-regulating system was found to be approximately 0, whereas the mean efflux was approximately 3.9 pmol X cm-2 X s-1. This net efflux required external HCO-3, external Na+, an acid pHi, internal ATP, and was blocked by SITS. We conclude that the pHi-regulating system mediates the obligate net influx of HCO-3 (or equivalent species) and Na+ and the net efflux of Cl- in the stoichiometry of 2:1:1. The transport system is stimulated by intracellular acid loads, requires ATP, and is blocked by SITS.

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