Glucocorticoids stimulate Na+/H+ antiporter in OKP cells

1993 ◽  
Vol 264 (6) ◽  
pp. F1027-F1031 ◽  
Author(s):  
M. Baum ◽  
A. Cano ◽  
R. J. Alpern
Keyword(s):  
Proximal Tubule ◽  
Intracellular Ph ◽  
Direct Effect ◽  
Initial Rate ◽  
Time Dependent ◽  
Affinity Constant ◽  
Maximal Velocity ◽  
Hemodynamic Changes ◽  
Acid Load ◽  
Stimulation Of

Previous studies have demonstrated that systemic administration of glucocorticoids stimulates proximal tubule acidification in part by increasing Na+/H+ antiporter activity; however, these studies could not exclude the possibility that changes in Na+/H+ antiporter activity were secondary to glucocorticoid-induced hemodynamic changes. The present study examined the effect of dexamethasone on Na+/H+ antiporter activity in quiescent OKP cells. Na+/H+ antiporter activity was assayed as the initial rate of Na(+)-dependent pH recovery from an acid load. Intracellular pH was measured using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Dexamethasone produced a dose- and time-dependent stimulation of Na+/H+ antiporter activity in OKP cells. Dexamethasone produced a 24% stimulation in Na+/H+ antiporter activity at 10(-9) M and an approximately 40% stimulation of Na+/H+ antiporter activity at both 10(-8) and 10(-6) M. The effect of 10(-6) M dexamethasone was seen within 4 h of incubation and was due to an increase in maximal velocity (Vmax, 3.03 vs. 1.79 pH units/min) with no change in the affinity constant for sodium (KNa, 47.2 vs. 42.0 mM). The stimulatory effect of dexamethasone on Na+/H+ antiporter activity was blocked by cycloheximide and was not observed with 10(-8) M aldosterone. These data demonstrate a direct effect of glucocorticoids to stimulate Na+/H+ antiporter activity in OKP cells.

Acid extrusion in S3 segment of rabbit proximal tubule. I. Effect of bilateral CO2/HCO3-

1995 ◽  
Vol 268 (2) ◽  
pp. F179-F192 ◽  
Author(s):  
L. K. Chen ◽  
W. F. Boron
Keyword(s):  
Steady State ◽  
Proximal Tubule ◽  
Initial Rate ◽  
Iodoacetic Acid ◽  
Buffering Power ◽  
Acid Load ◽  
S3 Segment ◽  
Series Of Experiments ◽  
Acid Extrusion ◽  
Stimulation Of

Monitoring the absorbance spectra of the pH-sensitive dye dimethylcarboxyfluorescein, we studied intracellular pH (pHi) regulation in the isolated perfused S3 segment of rabbit proximal tubule. To explain a previous observation, that steady-state pHi is higher in the presence than in the absence of CO2/HCO3- (N. L. Nakhoul, L. K. Chen, and W. F. Boron. J. Gen. Physiol. 102: 1171-1205, 1993), we examined the effect of bilateral (i.e., luminal and basolateral) CO2/HCO3- on the acid extrusion processes responsible for recovery of pHi from acid loads. To compute fluxes from rates of pHi change, we determined the pHi dependence of intrinsic intracellular buffering power, which was approximately 50 mM/pH at pHi 6.5 and fell linearly to approximately 20 mM at pHi 7.4. In one series of experiments, we monitored the rate of pHi recovery from an acid load imposed by an NH4+/NH3 prepulse. Over a broad range of pHi values, total net acid extrusion was approximately four times higher in bilateral presence of CO2/HCO3- than in its absence. In a second group of experiments, which were designed to determine the effect of CO2/HCO3- on luminal Na+/H+ exchange, we monitored the rate of pHi recovery elicited by adding Na+ back to only the lumen, after first removing Na+ bilaterally. Initial rate of luminal Na(+)-dependent net acid extrusion in presence of CO2/HCO3- was approximately 229 microM/s (pHi 6.92), approximately 1.8 times higher than the flux of approximately 127 microM/s (P < 0.005) obtained in absence of CO2/HCO3- (pHi 6.66). CO2/HCO3- alkali-shifted the flux vs. pHi relationship by 0.3-0.4 pH units. In a final series of experiments, we examined the effect of CO2/HCO3- on the Na(+)-independent alkalinization that follows the rapid, initial acidification elicited by bilateral Na+ removal. In the presence of CO2/HCO3-, lag time for initiation of the Na(+)-independent alkalinization was only approximately 36 vs. approximately 211 s (P < 0.002) in absence of CO2/HCO3-. Also, Na(+)-independent net acid extrusion rate was approximately two to three times higher in presence than in absence of CO2/HCO3- at comparable pHi. This Na(+)-independent acid extrusion was insensitive to N-ethylmaleimide (2 mM), but was inhibited approximately 94% by efforts to deplete intracellular ATP (i.e., removal of glucose and amino acids, plus addition of 2 mM cyanide and 10 mM iodoacetic acid). Stimulation of luminal Na+/H+ exchange and Na(+)-independent acid extrusion appears to be the major, if not the entire, explanation for the higher steady-state pHi caused by bilateral addition of CO2/HCO3-.

Stimulation of phosphate transport in the proximal tubule by metabolic substrates

1984 ◽  
Vol 247 (4) ◽  
pp. F582-F587 ◽  
Author(s):  
S. R. Gullans ◽  
P. C. Brazy ◽  
L. J. Mandel ◽  
V. W. Dennis
Keyword(s):  
Proximal Tubule ◽  
Fluid Transport ◽  
Initial Rate ◽  
Tricarboxylic Acid Cycle ◽  
Phosphate Transport ◽  
Glucose Absorption ◽  
Metabolic Substrates ◽  
Tricarboxylic Acid Cycle Intermediates ◽  
Stimulation Of

Studies of phosphate transport in the proximal tubule have recently focused on interactions with cellular metabolism. The present studies demonstrate that two fatty acids, valerate and butyrate, and two tricarboxylic acid cycle intermediates, succinate and malate, stimulate net phosphate transport in the rabbit proximal tubule by 34-117%. Valerate had no effect on the total uptake of inorganic [32P]phosphate into suspensions of proximal tubules but did enhance the initial rate of influx. Net fluid transport was unaffected by these substrates although glucose absorption increased by 10-15% following the addition of either valerate or succinate. Since valerate, butyrate, and succinate are known to stimulate gluconeogenesis and respiration, we evaluated the role of gluconeogenesis in the stimulation of phosphate transport. The addition of 3-mercaptopicolinate (1 mM), an inhibitor of gluconeogenesis, did not alter phosphate transport, nor did it prevent the valerate-induced stimulation of phosphate transport. We conclude that valerate, butyrate, succinate, and malate enhance phosphate transport by the proximal convoluted tubule. This action appears to be unrelated to effects on gluconeogenesis and may be related to close links between phosphate transport and oxidative metabolism.

Regulation of intracellular pH in J774 murine macrophage cells: H+ extrusion processes

1995 ◽  
Vol 268 (1) ◽  
pp. C210-C217 ◽  
Author(s):  
L. C. McKinney ◽  
A. Moran
Keyword(s):  
Intracellular Ph ◽  
Initial Rate ◽  
Adherent Cells ◽  
Murine Macrophage ◽  
Bafilomycin A1 ◽  
Macrophage Cell ◽  
Recovery From Acidification ◽  
J774 Cells ◽  
Acid Load ◽  
Rate Of Recovery

Mechanisms of intracellular pH (pHi) regulation were characterized in the murine macrophage cell line J774.1, using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to measure pHi. Under nominally HCO3(-)-free conditions, resting pHi of nonadherent J774.1 cells was 7.53 +/- 0.02 (n = 86), and of adherent cells was 7.59 +/- 0.02 (n = 97). In the presence of HCO3-/CO2, pHi values were reduced to 7.41 +/- 0.02 (n = 12) and 7.40 +/- 0.01 (n = 28), respectively. Amiloride, an inhibitor of Na+/H+ exchange, did not affect resting pHi. Inhibitors of a vacuolar type H(+)-ATPase [bafilomycin A1, N-ethylmaleimide (NEM), 7-chloro-4-nitrobenz-2-oxa-1,3-diazide (NBD), and p-chloromercuriphenylsulfonic acid (pCMBS)] reduced pHi by at least 0.2 pH units. Inhibitors of other classes of H(+)-ATPases (oligomycin, azide, vanadate, and ouabain) were without effect. Inhibition of H+ efflux, measured by the change in extracellular pH of a weakly buffered cell suspension, followed the same pharmacological profile, indicating that the reduction of pHi was due to inhibition of H+ extrusion. Mechanisms of recovery from an imposed intracellular acid load were also investigated. In NaCl-Hanks' solution, pHi recovered exponentially to normal within 2 min. The initial rate of recovery was inhibited > 90% by amiloride or by replacement of extracellular Na+ concentration by N-methyl-glucamine. Inhibitors of the vacuolar H(+)-ATPase also inhibited recovery. NEM and NBD nonspecifically inhibited all recovery. Bafilomycin A1 and pCMBS did not inhibit the initial amiloride-sensitive portion of recovery, but they did inhibit a late component of recovery when pHi was above 7.0. We conclude that the Na+/H+ exchanger is primarily responsible for recovery from an acid load but does not regulate resting pHi. Conversely, a vacuolar H(+)-ATPase regulates the resting pHi of J774 cells but contributes little to recovery from acidification.

Intracellular pH regulation in rabbit S3 proximal tubule: basolateral Cl-HCO3 exchange and Na-HCO3 cotransport

1990 ◽  
Vol 258 (2) ◽  
pp. F371-F381 ◽  
Author(s):  
N. L. Nakhoul ◽  
L. K. Chen ◽  
W. F. Boron
Keyword(s):  
Proximal Tubule ◽  
Intracellular Ph ◽  
Initial Rate ◽  
Ph Regulation ◽  
Basolateral Membrane ◽  
Intracellular Ph Regulation ◽  
S3 Segment ◽  
Absorbance Spectra ◽  
Rule Out

We studied the role of basolateral HCO3- transport in the regulation of intracellular pH (pHi) in the isolated perfused S3 segment of the rabbit proximal tubule. pHi was calculated from absorbance spectra of the pH-sensitive dye dimethylcarboxyfluorescein. Solutions were normally buffered to pH 7.4 at 37 degrees C with 25 mM HCO3- 5% CO2. pHi fell by approximately 0.17 when luminal [HCO3-] was lowered to 5 mM at fixed PCO2 (i.e., reducing pH to 6.8) but by approximately 0.42 when [HCO3-] in the bath (i.e., basolateral solution) was lowered to 5 mM. The pHi decrease elicited by reducing bath [HCO3-] was substantially reduced by removal of Cl- or Na+, suggesting that components of basolateral HCO3- transport are Cl- and/or Na+ dependent. We tested for the presence of basolateral Cl-HCO3 exchange by removing bath Cl-. This caused pHi to increase by approximately 0.23, with an initial rate of approximately 100 X 10(-4) pH/s. Although the initial rate of this pHi increase was not reduced by removing Na+ bilaterally, it was substantially lowered by the nominal removal of HCO3- from bath and lumen or by the addition of 0.1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) to the bath. The results thus suggest that a Na-independent Cl-HCO3 exchanger is present at the basolateral membrane. We tested for the presence of basolateral Na-HCO3 cotransport by removing bath Na+. This caused pHi to fall reversibly by approximately 0.26 with initial rates of pHi decline and recovery being approximately 30 and approximately 41 X 10(-4) pH/s, respectively. Although the bilateral removal of Cl- had no effect on these rates, the nominal removal of HCO3- or the presence of DIDS substantially slowed the pHi changes. Thus, in addition to a Cl-HCO3 exchanger, the basolateral membrane of the S3 proximal tubule also appears to possess a Na-HCO3 cotransport mechanism. The data do not rule out the possibility of other basolateral HCO3- transporters.

Hyperosmotic mannitol activates basolateral NHE in proximal tubule from P-glycoprotein null mice

2002 ◽  
Vol 282 (4) ◽  
pp. F718-F729 ◽  
Author(s):  
Yukio Miyata ◽  
Yasushi Asano ◽  
Shigeaki Muto
Keyword(s):  
Proximal Tubule ◽  
Maximal Velocity ◽  
Wild Type ◽  
Acetoxymethyl Ester ◽  
Calphostin C ◽  
Kinase C ◽  
P Glycoprotein ◽  
Acid Load ◽  
Acid Extrusion ◽  
Menten Constant

Using the pH-sensitive fluorescent dye 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester, we examined the effects of hyperosmotic mannitol on basolateral Na+/H+ exchange (NHE) activity in isolated nonperfused proximal tubule S2 segments from mice lacking both the mdr1a and mdr1b genes (KO) and wild-type mice (WT). All experiments were performed in CO2/HCO[Formula: see text]-free HEPES solutions. Osmolality of the peritubular solution was raised from 300 to 500 mosmol/kgH2O by the addition of mannitol. NHE activity was assessed by Na+-dependent acid extrusion rates ( J H) after an acid load with NH4Cl prepulse. Under isosmotic conditions, J H values at a wide intracellular pH (pHi) range of 6.20–6.90 were not different between the two groups. In WT mice, hyperosmotic mannitol had no effect on J H at the wide pHi range. In contrast, in KO mice, hyperosmotic mannitol increased J H at a pHi range of 6.20–6.45 and shifted the J H-pHi relationship by 0.15 pH units in the alkaline direction. In KO mice, hyperosmotic mannitol caused an increase in maximal velocity without changing the Michaelis-Menten constant for peritubular Na+. Exposure of cells from WT mice to the hyperosmotic mannitol solution including the P-gp inhibitor cyclosporin A increased J H (at pHi6.30) to an extent similar to that in cells from KO mice exposed to hyperosmotic mannitol alone. In KO mice, staurosporine and calphostin C inhibited the hyperosmotic mannitol-induced increase in J H. The stimulatory effect of hyperosmotic mannitol on J H was mimicked by addition to the isosmotic control solution, including phorbol 12-myristate 13-acetate (PMA; the PKC activator). In WT mice, hyperosmotic mannitol with PMA increased J H. We conclude that, in the absence of P-gp activity, hyperosmotic mannitol activates basolateral NHE via protein kinase C, whereas in the presence of P-gp activity, it does not.

LLC-PK1 mutant with increased Na+-H+ exchange and decreased sensitivity to amiloride

1988 ◽  
Vol 255 (4) ◽  
pp. C495-C501 ◽  
Author(s):  
J. G. Haggerty ◽  
N. Agarwal ◽  
E. J. Cragoe ◽  
E. A. Adelberg ◽  
C. W. Slayman
Keyword(s):  
Amino Acid ◽  
Intracellular Ph ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Maximal Velocity ◽  
Detectable Change ◽  
Inhibitory Potency ◽  
Acid Load ◽  
22Na Uptake ◽  
Na Uptake

LLC-PK1 cells contain a well-characterized Na+-H+ antiporter that is sensitive to ethylisopropylamiloride (EIPA) in the submicromolar range. Using a modification of the method of Franchi et al. (J. Biol. Chem. 261: 14614-14620, 1986), we have selected mutants that can recover from an acid load in the presence of 100 microM EIPA. One such mutant, designated PKE20, has been studied in detail. The maximal velocity (Vmax) for the Na+-H+ antiporter, assayed as EIPA-sensitive 22Na+ uptake, has increased from 44 nmol.min-1.10(6) cells-1 in the parent cells to 106 nmol.min-1.10(6) cells-1 in PKE20. No detectable change has occurred in the Km for Na+ (118 mM in the parent, 111 mM in the mutant) or in the dependence of Na+ uptake on intracellular pH. However, the PKE20 antiporter exhibits a greatly decreased sensitivity to amiloride and its derivatives, with drops in inhibitory potency ranging from 25-fold (amiloride) to 100-fold (EIPA). The mutation is specific for the antiporter; measurements of Na+-K+ pump and Na+-dependent amino acid uptake show only small changes, which appear to result from minor antiporter-induced alterations in internal Na+ concentration. PKE20 cells should prove useful in experiments to identify and isolate the antiporter protein.

Na+/H+ exchange in mosquito Malpighian tubules

2000 ◽  
Vol 279 (6) ◽  
pp. R1996-R2003 ◽  
Author(s):  
D. H. Petzel
Keyword(s):  
Steady State ◽  
Intracellular Ph ◽  
Specific Inhibitor ◽  
Fluid Secretion ◽  
Malpighian Tubules ◽  
Pulse Technique ◽  
Acid Load ◽  
Rate Of Recovery ◽  
Stimulation Of

Fluid secretion and intracellular pH were measured in isolated mosquito Malpighian tubules to determine the presence of Na+/H+ exchange. Rates of fluid secretion by individual Malpighian tubules in vitro were inhibited by 78% of control in the presence of 100 μM 5-( N-ethyl- n-isopropyl)-amiloride (EIPA), a specific inhibitor of Na+/H+ exchange. Steady-state intracellular pH was measured microfluorometrically by using 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in individual Malpighian tubules. Bathing the Malpighian tubules in 0 mM extracellular Na+ or in the presence of 100 μM EIPA reduced the steady-state intracellular pH by 0.5 pH units. Stimulation of the Na+/H+ exchanger by using the NH4Cl pulse technique resulted in a rate of recovery from the NH4Cl-induced acute acid load of 8.7 ± 1.0 × 10−3 pH/s. The rates of recovery of intracellular pH after the acute acid load in the absence of extracellular Na+ or in the presence of 100 μM EIPA were 0.7 ± 0.6 and −0.3 ± 0.3 × 10−3 pH/s, respectively. These results indicate that mosquito Malpighian tubules possess a Na+/H+ exchanger.

scholarly journals Intracellular pH regulation in the S3 segment of the rabbit proximal tubule in HCO3- -free solutions.

1988 ◽  
Vol 92 (3) ◽  
pp. 369-393 ◽  
Author(s):  
N L Nakhoul ◽  
A G Lopes ◽  
J R Chaillet ◽  
W F Boron
Keyword(s):  
Proximal Tubule ◽  
Intracellular Ph ◽  
Ph Regulation ◽  
Transport Systems ◽  
Total Acid ◽  
Luminal Membrane ◽  
Acid Load ◽  
S3 Segment ◽  
Acid Extrusion ◽  
Dependent Mechanism

We used the absorbance spectrum of 4',5'-dimethyl-5-(and 6) carboxyfluorescein to measure intracellular pH (pHi) in the isolated, perfused S3 segment of the rabbit proximal tubule. Experiments were conducted in HCO3- -free solutions. pHi recovered from an acid load imposed by an NH4+ prepulse, indicating the presence of one or more active acid-extrusion mechanisms. Removal of Na+ from bath and lumen caused pHi to decrease by approximately 0.6, whereas Na+ readdition caused complete pHi recovery. Removal of Na+ from the bath caused only a slow pHi decrease that was enhanced about fourfold when Na+ was subsequently removed from the lumen also. Similarly, the pHi recovery produced by the readdition of Na+ to the bath and lumen was about ninefold faster than when Na+ was returned to the bath only. Amiloride (1-2 mM) inhibited the pHi recovery that was elicited by returning 15 or 29 mM Na+ to lumen by only approximately 30%. However, in the absence of external acetate (Ac-), 1 mM amiloride inhibited approximately 66% of the pHi recovery induced by the readdition of 29 mM Na+ to the lumen only. The removal of external Ac- reduced the pHi recovery rate from an NH4+-induced acid load by approximately 47%, and that elicited by Na+ readdition, by approximately 67%. Finally, when bilateral removal of Na+ was maintained for several minutes, pHi recovered from the initial acidification, slowly at first, and then more rapidly, eventually reaching a pHi approximately 0.1 higher than the initial one. This Na+-independent pHi recovery was not significantly affected by lowering [HEPES]o from 32 to 3 mM or by adding N'N'-dicyclohexylcarbodiimide (10(-4) M) to the lumen, but it was reduced approximately 57% by iodoacetate (0.5 mM) plus cyanide (1 mM). We conclude that in the nominal absence of HCO3-, three transport systems contribute to acid extrusion by S3 cells: (a) a Na+-independent mechanism, possibly an H+ pump; (b) a Na-H exchanger, confined primarily to the luminal membrane; and (c) an Ac- and luminal Na+-dependent mechanism. The contribution of these three mechanisms to total acid extrusion, assessed by the rapid readdition of Na+, was approximately 13, approximately 30, and approximately 57%, respectively.

Effects of pH on basolateral tetraethylammonium transport in snake renal proximal tubules

1997 ◽  
Vol 272 (3) ◽  
pp. R955-R961 ◽  
Author(s):  
Y. K. Kim ◽  
W. H. Dantzler
Keyword(s):  
Membrane Potential ◽  
Intracellular Ph ◽  
Direct Effect ◽  
Basolateral Membrane ◽  
Extracellular Ph ◽  
Proximal Tubules ◽  
Renal Proximal Tubules ◽  
Effects Of Ph ◽  
The One ◽  
Stimulation Of

Previous work on snake renal proximal tubules suggested that pH might influence tetraethylammonium (TEA) transport across the basolateral membrane. To examine this more directly, we determined the effects of altering either extracellular pH (pHo) or intracellular pH (pHi) on TEA uptake and efflux across the basolateral membrane of isolated snake renal proximal tubules. We found no evidence for trans-stimulation of either TEA uptake or efflux by H+. Therefore, there was no evidence for a TEA/H+ exchanger. However, we found evidence for trans-inhibition of both TEA uptake and efflux as well as for cis-inhibition of TEA uptake by increasing H+ concentration. H+ concentration appeared to have some type of direct effect on basolateral transport independent of any effect on membrane potential. Moreover, there appeared to be an optimal intracellular H+ concentration for entry of TEA into the cells that corresponded to the one found at the physiological pHi of 7.1. There also appeared to be an optimal extracellular H+ concentration for efflux of TEA from the cells that corresponded to the one found at the physiological pHo of 7.4. The mechanism involved in this relationship is unknown, but the data support a concept derived from previous studies that TEA transport across the basolateral membrane is asymmetric.

Effect of isolated removal of either basolateral HCO3-or basolateral CO2on HCO3-reabsorption by rabbit S2 proximal tubule

2003 ◽  
Vol 285 (2) ◽  
pp. F359-F369 ◽  
Author(s):  
Jinhua Zhao ◽  
Yuehan Zhou ◽  
Walter F. Boron
Keyword(s):  
Proximal Tubule ◽  
Intracellular Ph ◽  
Proximal Tubules ◽  
Bath Solution ◽  
Competing Hypotheses ◽  
Stimulation Of ◽  
Minimal Formula

The equilibrium [Formula: see text] had made it impossible to determine how isolated changes in basolateral CO2([CO2]) or [Formula: see text] concentration ([[Formula: see text]]), at a fixed basolateral pH, modulate renal [Formula: see text] or reabsorption. In the present study, we have begun to address this issue by measuring [Formula: see text] reabsorption ( JHCO3) and intracellular pH (pHi) in isolated perfused rabbit S2 proximal tubules exposed to three different basolateral (bath) solutions: 1) equilibrated 5% CO2/22 mM [Formula: see text] 7.40, 2) an out-of-equilibrium (OOE) solution containing 5% CO2/pH 7.40 but minimal [Formula: see text] (“pure CO2”), and 3) an OOE solution containing 22 mM [Formula: see text] 7.40 but minimal CO2(“pure [Formula: see text]”). Tubule lumens were constantly perfused with equilibrated 5% CO2/22 mM [Formula: see text]. Compared with the equilibrated bath solution ( JHCO3= 76.5 ± 7.7 pmol·min–1·mm–1, pHi= 7.09 ± 0.04), the pure CO2bath solution increased JHCO3by ∼25% but decreased pHiby 0.19. In contrast, the pure [Formula: see text] bath solution decreased JHCO3by 37% but increased pHiby 0.24. Our data are consistent with two competing hypotheses: 1) the isolated removal of basolateral [Formula: see text] (or CO2) causes a pHidecrease (increase) that in turn raises (lowers) JHCO3; and 2) [Formula: see text] removal raises JHCO3by reducing inhibition of basolateral Na/HCO3cotransport and/or reducing [Formula: see text] backleak, whereas CO2removal lowers JHCO3by reducing stimulation of a CO2sensor.

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