scholarly journals Further studies of the volume-regulatory response of Amphiuma red cells in hypertonic media. Evidence for amiloride-sensitive Na/H exchange.

1985 ◽  
Vol 86 (4) ◽  
pp. 565-584 ◽  
Author(s):  
F M Kregenow ◽  
T Caryk ◽  
A W Siebens
Keyword(s):  
Anion Exchanger ◽  
Red Cells ◽  
Disulfonic Acid ◽  
Medium Ph ◽  
Previous Evidence ◽  
Medium Acidification ◽  
Initial Event ◽  
Na Ions ◽  
Volume Regulatory Increase ◽  
Na Uptake

When Amphiuma red cells are shrunken in hypertonic media, they return toward their original volume by gaining Na through an amiloride-sensitive pathway. As cells recover their volume during this volume-regulatory increase (VRI) response, acid is extruded into the medium. Medium acidification is correlated with cell Na uptake. Both medium acidification and cell Na uptake are blocked by 10(-3) M amiloride or by replacing medium Na with K or choline. Perturbations that increase cell Na uptake (such as increasing medium osmolality) also increase medium acidification. As the medium becomes more acidic, the cells become more alkaline. These changes in cell and medium pH are increased if pH equilibration across the cell membrane is prevented by inhibiting the anion exchanger with SITS (4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonic acid). The quantity of acid extruded by SITS-treated cells is the same as the quantity of Na gained, which strongly suggests 1:1 exchange of Na for H. Cell enlargement in SITS-treated cells results from the exchange of osmotically active Na ions for H ions that are not osmotically active when combined with cellular buffers. Previous evidence indicates that the normal VRI response involves an increase in the cellular content of Cl as well as Na. We show that SITS completely blocks net Cl uptake, which suggests that Cl enters via the anion exchanger. SITS also slows Na entry, presumably as a result of the above-mentioned increase in cell pH caused by SITS. We suggest that the initial event in the VRI response is net Na uptake via a Na/H exchanger, and that net Cl uptake results from secondary Cl/HCO3 exchange via the anion exchanger.

scholarly journals Volume-regulatory responses of Amphiuma red cells in anisotonic media. The effect of amiloride.

1985 ◽  
Vol 86 (4) ◽  
pp. 527-564 ◽  
Author(s):  
A W Siebens ◽  
F M Kregenow
Keyword(s):  
Fold Increase ◽  
Competitive Inhibitor ◽  
Red Cells ◽  
Salt Transport ◽  
Transport Mechanisms ◽  
Volume Regulatory Decrease ◽  
Pump Activity ◽  
Original Size ◽  
Volume Regulatory Increase ◽  
Na Uptake

Amphiuma red cells were incubated for several hours in hypotonic or hypertonic media. They regulate their volume in both media by using ouabain-insensitive salt transport mechanisms. After initially enlarging osmotically, cells in hypotonic media return toward their original size by losing K, Cl, and H2O. During this volume-regulatory decrease (VRD) response, K loss results from a greater than 10-fold increase in K efflux. Cells in hypertonic media initially shrink osmotically, but then return toward their original volume by gaining Na, Cl, and H2O. The volume-regulatory increase (VRI) response involves a large (greater than 100-fold) increase in Na uptake that is entirely blocked by the diuretic amiloride (10(-3) M). Na transport in the VRI response shares many of the characteristics of amiloride-sensitive transport in epithelia: (a) amiloride inhibition is reversible; (b) removal of amiloride from cells pretreated with amiloride enhances Na uptake relative to untreated controls; (c) amiloride appears to act as a competitive inhibitor (Ki = 1-3 microM) of Na uptake; (d) Na uptake is a saturable function of external Na (Km approximately 29 mM); (e) Li can substitute for Na but K cannot. Anomalous Na/K pump behavior is observed in both the VRD and the VRI responses. In the VRD response, pump activity increases 3-fold despite a decrease in intracellular Na concentration, while in the VRI response, a 10-fold increase in pump activity is observed when only a doubling is predicted from increases in intracellular Na.

scholarly journals Characterization of anion exchangers in an inner medullary collecting duct cell line.

1998 ◽  
Vol 9 (5) ◽  
pp. 746-754
Author(s):  
G Obrador ◽  
H Yuan ◽  
T M Shih ◽  
Y H Wang ◽  
M A Shia ◽  
...  
Keyword(s):  
Cell Line ◽  
Anion Exchanger ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Disulfonic Acid ◽  
Kidney Cortex ◽  
Intercalated Cells ◽  
Rat Stomach ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct

Although the inner medullary collecting duct (IMCD) plays a major role in urinary acidification, the molecular identification of many of the specific components of the transport system in this nephron segment are lacking. A cultured line of rat IMCD cells was used to characterize the mediators of cellular HCO3 exit. This cell line functionally resembles alpha-intercalated cells. Physiologic experiments document that HCO3- transport is a reversible, electroneutral, Cl dependent, Na+-independent process. It can be driven by Cl-gradients and inhibited by stilbenes such as 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid. Immunohistochemical analysis, using a rabbit polyclonal antibody against the carboxy-terminal 12 amino acids of anion exchanger 1 (AE1), revealed a distribution of immunoreactive protein that is consistent with a basolateral localization of AE in cultured cells and in alpha-intercalated cells identified in sections of rat kidney cortex. Immunoblot revealed two immunoreactive bands (approximately 100 and 180 kD in size) in membranes from cultured IMCD cells, rat renal medulla, and freshly isolated IMCD cells. The mobility of the lower molecular weight band was similar to that of AE1 in red blood cell ghosts and kidney homogenate and therefore probably represents AE1. The mobility of the 180-kD band is similar to that for rat stomach and kidney AE2 and therefore probably represents AE2. Selective biotinylation of the apical or basolateral membrane proteins in cultured IMCD cells revealed that both AE1 and AE2 are polarized to the basolateral membrane. Northern blot analysis documented the expression of mRNA for AE1 and AE2 but not AE3. Furthermore, the cDNA sequence of AE1 and AE2 expressed by these cells was found to be virtually identical to that reported for kidney AE1 and rat stomach AE2. It is concluded that this cultured line of rat IMCD cells expresses two members of the anion exchanger gene family, AE1 and AE2, and both of these exchangers probably mediate the electroneutral Cl--dependent HCO3-transport observed in this cell line.

scholarly journals Methylprednisolone inhibits uptake of Ca2+ and Na+ ions into concanavalin A-stimulated thymocytes

1997 ◽  
Vol 326 (2) ◽  
pp. 329-332 ◽  
Author(s):  
Frank BUTTGEREIT ◽  
Stefan KRAUSS ◽  
Martin D. BRAND
Keyword(s):  
Plasma Membrane ◽  
Concanavalin A ◽  
Membrane Properties ◽  
Mitochondrial Electron Transport Chain ◽  
Oxidation Reactions ◽  
Transport Chain ◽  
Cord Injury ◽  
Na Ions ◽  
Atp Supply ◽  
Na Uptake

The glucocorticoid drug methylprednisolone inhibits respiration in concanavalin A-stimulated rat thymocytes at concentrations that are relevant to its acute clinical efficacy against autoimmune diseases and spinal cord injury. Methylprednisolone affects several processes, including ion cycling, substrate oxidation reactions and RNA/DNA synthesis. The inhibition of respiration used to drive ATP-consuming cycles of Ca2+ and Na+ ions across the plasma membrane has been proposed to be either primary or secondary to restriction of cellular ATP supply. By comparing the effects of methylprednisolone with those of myxothiazol, an inhibitor of the mitochondrial electron transport chain, we show that the effects of methylprednisolone on Ca2+ and Na+ cycling are primary. We propose that methylprednisolone acts by affecting membrane properties to inhibit Ca2+ and Na+ uptake across the plasma membrane and to increase H+ uptake across the mitochondrial membrane, and that other effects are secondary.

Sodium entry mechanisms in distal convoluted tubule cells

1995 ◽  
Vol 268 (1) ◽  
pp. F89-F98 ◽  
Author(s):  
F. A. Gesek ◽  
P. A. Friedman
Keyword(s):  
Competitive Inhibitor ◽  
Distal Convoluted Tubule ◽  
Hill Coefficient ◽  
Disulfonic Acid ◽  
Phosphonoformic Acid ◽  
Exchange Inhibitor ◽  
Michaelis Constants ◽  
Tubule Cells ◽  
Apical Membranes ◽  
Na Uptake

Sodium transport across apical membranes of distal convoluted tubules is thought to be mediated by Na-Cl cotransport and conductive Na entry. Immortalized mouse distal convoluted tubule cells were used to characterize Na entry pathways. Chlorothiazide, an inhibitor of Na-Cl cotransport, and amiloride, which blocks epithelial Na channels, reduced ouabain-suppressible oxygen consumption by 40 and 35%, respectively. In simple buffer solutions, free of bicarbonate, phosphate, or formate, chlorothiazide inhibited Na uptake by 44% and Cl uptake by 48%. Michaelis constants of 21 mM for Na and 14 mM for chloride were calculated. Amiloride inhibited Na uptake by 49% and had no effect on Cl uptake. The calculated Hill coefficient of 1.07 and the equivalence of chlorothiazide-sensitive Na and Cl uptake are consistent with the presence of Na-Cl cotransport. Na-Cl cotransport and amiloride-sensitive Na influx account for 85% of Na entry in distal convoluted tubule cells in the absence of phosphate and formate. The selective Na/H exchange inhibitor ethylisopropyl amiloride had no effect on Na uptake; however, it abolished formate-stimulated Na uptake. The anion exchange blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited Na uptake. These findings are consistent with parallel Na/H and Cl/formate exchange. Na uptake was inhibited 8% by the selective Na/Ca exchange inhibitor, dimethylbenzamil. An additional 7% of Na entry was phosphate dependent and was abolished by phosphonoformic acid, a competitive inhibitor of Na-Pi cotransport. In summary, the majority of Na entry into distal convoluted tubule cells occurs through Na-Cl cotransport and an amiloride-sensitive pathway (75% in presence of phosphate and formate). An additional 13% may enter by Na/H exchange, with the remainder mediated by Na/Ca exchange and Na-Pi cotransport.

Cellular actions of cAMP on HCO3(-)-secreting cells of rabbit CCD: dependence on in vivo acid-base status

1994 ◽  
Vol 266 (4) ◽  
pp. F528-F535 ◽  
Author(s):  
C. Emmons ◽  
J. B. Stokes
Keyword(s):  
Anion Exchanger ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Disulfonic Acid ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Acid Base ◽  
Acid Base Status

HCO3- secretion by cortical collecting duct (CCD) occurs via beta-intercalated cells. In vitro CCD HCO3- secretion is modulated by both the in vivo acid-base status of the animal and by adenosine 3',5'-cyclic monophosphate (cAMP). To investigate the mechanism of cAMP-induced HCO3- secretion, we measured intracellular pH (pHi) of individual beta-intercalated cells of CCDs dissected from alkali-loaded rabbits perfused in vitro. beta-Intercalated cells were identified by demonstrating the presence of an apical anion exchanger (cell alkalinization in response to removal of lumen Cl-). After 180 min of perfusion to permit decrease of endogenous cAMP, acute addition of 0.1 mM 8-bromo-cAMP or 1 microM isoproterenol to the bath caused a transient cellular alkalinization (> 0.20 pH units). In the symmetrical absence of either Na+, HCO3-, or Cl-, cAMP produced no change in pHi. Basolateral dihydrogen 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (0.1 mM) for 15 min before cAMP addition also prevented this alkalinization. In contrast to the response of cells from alkali-loaded rabbits, addition of basolateral cAMP to CCDs dissected from normal rabbits resulted in an acidification of beta-intercalated cells (approximately 0.20 pH units). The present studies demonstrate the importance of the in vivo acid-base status of the animal in the regulation of CCD HCO3- secretion by beta-intercalated cells. The results identify the possible existence of a previously unrecognized Na(+)-dependent Cl-/HCO3- exchanger on the basolateral membrane of beta-intercalated cells in alkali-loaded rabbits.

Functional characterization and regulation by pH of murine AE2 anion exchanger expressed in Xenopus oocytes

1994 ◽  
Vol 267 (5) ◽  
pp. C1295-C1307 ◽  
Author(s):  
B. D. Humphreys ◽  
L. Jiang ◽  
M. N. Chernova ◽  
S. L. Alper
Keyword(s):  
Anion Exchanger ◽  
Transport Mechanism ◽  
Xenopus Oocytes ◽  
Rank Order ◽  
Functional Characterization ◽  
Disulfonic Acid ◽  
Transport Function ◽  
Related Protein ◽  
Nitrate Medium ◽  
Free Media

cRNA encoding the murine band 3-related protein AE2 was expressed in Xenopus oocytes. AE2-mediated transport function and regulation were analyzed by unidirectional 36Cl- influx and efflux studies. AE2 cRNA-injected oocytes took up 36Cl- as much as 40-fold faster than did water-injected oocytes. AE2-mediated 36Cl- uptake increased as a function of increasing uptake time, number of days after cRNA injection, and amount of injected cRNA. Among the functional properties of AE2 evaluated were transport mechanism and substrate specificity, inhibitor pharmacology, and regulation by pH. The apparent Km for external Cl- was 5.6 mM. AE2 was defined as a Cl-/anion exchanger by two criteria: 1) 36Cl- efflux from AE2-expressing oocytes was maximally stimulated by extracellular Cl- or nitrate; AE2-associated 36Cl- efflux was supported by substitution of extracellular Cl- with other anions in the rank order bromide > isethionate > or = gluconate > iodide and 2) prolonged preincubation of AE2 cRNA-injected oocytes in Cl(-)-free media containing isethionate, gluconate, or glutamate decreased subsequent AE2-associated 36Cl- uptake from Cl- media in rough proportion to the degree of intracellular Cl- depletion, whereas preincubation in nitrate medium had no effect. AE2-associated 36Cl- uptake was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid at half-maximally inhibitory concentrations between 0.5 and 19 microM, depending on extracellular Cl- concentration, and progressed to irreversibility at 20 degrees C with a half-time of 20-30 min. Many additional inhibitors showed lower potency for AE2 than previously reported for AE1. Although AE2 expression did not change oocyte resting intracellular pH, AE2-associated 36Cl- influx and efflux were each decreased in acid incubation medium and increased in alkaline medium.

Stimulus-secretion coupling in beta-cells: modulation by pH

1983 ◽  
Vol 244 (1) ◽  
pp. E3-E18 ◽  
Author(s):  
C. S. Pace ◽  
J. T. Tarvin ◽  
J. S. Smith
Keyword(s):  
Beta Cell ◽  
Spike Activity ◽  
Active Phase ◽  
Secretory Process ◽  
Disulfonic Acid ◽  
Medium Ph ◽  
Oscillatory Pattern ◽  
Stimulus Secretion Coupling ◽  
Influence Of Ph ◽  
K Permeability

We have examined the influence of changes in pH on the oscillatory pattern of electrical activity (EA) in the beta-cell by altering medium pH (pHo) and using permeable weak buffers to alter intracellular pH (pHi). A decrease in pH in the presence of glucose elicited depolarization to the active phase and constant spike activity, whereas an increase in pH elicited a decrease in spike activity or silent hyperpolarization. On inhibition of HCO3:Cl antiport by addition of DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonic acid), probenecid, or withdrawal of medium HCO-3, there was an increase in the duration of the active phase. A similar result was obtained on the inhibition of Na:H antiport by the addition of amiloride or the reduction of medium [Na+]. The influence of H+ and glucose has been proposed to decrease K+ permeability (PK). However, the influence of pH on 86Rb+ efflux was most effective at subthreshold or 4.2 mM glucose; only a moderate decrease in PK occurred at 8.3 mM glucose, and no effect was obtained at 16.7 mM glucose. Alteration of pHi, and not pHo, induces similar effects on glucose-induced electrical and secretory events. There is a clear dissociation between the influence of inhibitors of the Na:H and HCO3:Cl antiporters on the electrical and secretory events. DIDS and amiloride increased glucose-induced EA, but markedly inhibited the secretory response to glucose. It is evident that pH modulates the electrical events and cationic fluxes and ultimately influences the transduction of information to the mechanisms controlling the secretory process in the beta-cell.

Na-H exchange in rat liver basolateral but not canalicular plasma membrane vesicles

1986 ◽  
Vol 250 (1) ◽  
pp. G35-G43 ◽  
Author(s):  
R. H. Moseley ◽  
P. J. Meier ◽  
P. S. Aronson ◽  
J. L. Boyer
Keyword(s):  
Rat Liver ◽  
Membrane Vesicles ◽  
Disulfonic Acid ◽  
Plasma Membrane Vesicles ◽  
Bile Formation ◽  
Proton Gradients ◽  
Ph Dependent ◽  
22Na Uptake ◽  
Proton Movement ◽  
Na Uptake

Na+-stimulated H+ movement and H+-stimulated Na+ uptake were studied in basolateral (blLPM) and canalicular (cLPM) rat liver membrane vesicles. H+ movement was monitored with the fluorescent amine acridine orange; 22Na uptake was assayed by a rapid Millipore filtration technique. In blLPM, inwardly directed Na+ gradients stimulated H+ efflux and outwardly directed Na+ gradients stimulated proton influx. Outwardly directed proton gradients (pH in 5.9/pH out 7.9) stimulated initial 22Na uptake rates 5- to 10-fold over pH-equilibrated conditions (pH in 7.9/pH out 7.9). Conversely, inwardly directed proton gradients (pH in 7.9/pH out 5.9) inhibited 22Na uptake. pH-dependent 22Na uptake was inhibited by amiloride and harmaline but not by other transport inhibitors, bumetanide, furosemide, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, and acetazolamide. Lithium also inhibited H+-stimulated 22Na uptake. Although a component of pH-stimulated 22Na uptake appeared to be dependent on membrane potential, this electrogenic component was amiloride insensitive. Proton gradient-stimulated 22Na uptake in blLPM was saturable, with a Km of 5.4 mM and a Vmax of 14 nmol . min-1 . mg prot-1. In contrast, in cLPM, no Na+ gradient-stimulated proton movement and no pH-dependent Na+ uptake occurred. These findings establish an electroneutral Na-H antiport in blLPM but not cLPM in rat liver. The polarity of this exchanger supports a model of bile formation that is dependent, in part, on canalicular HCO-3 and/or OH- excretion.

Characterization of apical membrane Cl-dependent Na/H exchange in crypt cells of rat distal colon

2001 ◽  
Vol 280 (3) ◽  
pp. G400-G405 ◽  
Author(s):  
Vazhaikkurichi M. Rajendran ◽  
John Geibel ◽  
Henry J. Binder
Keyword(s):  
Channel Blocker ◽  
Apical Membrane ◽  
Distal Colon ◽  
Proton Gradient ◽  
Disulfonic Acid ◽  
High Dose ◽  
Cl Channel ◽  
Rat Distal Colon ◽  
Na Uptake ◽  
Crypt Cells

A novel Cl-dependent Na/H exchange (Cl-NHE) has been identified in apical membranes of crypt cells of rat distal colon. The presence of Cl is required for both outward proton gradient-driven Na uptake in apical membrane vesicles (AMV) and Na-dependent intracellular pH recovery from an acid load in the crypt gland. The present study establishes that Cl-dependent outward proton gradient-driven 22Na uptake 1) is saturated with increasing extravesicular Na concentration with a Michaelis constant ( K m) for Na of ∼24.2 mM; 2) is saturated with increasing outward H concentration gradient with a hyperbolic curve and a K m for H of ∼1.5 μM; 3) is inhibited by the Na/H exchange (NHE) inhibitors amiloride, ethylisopropylamiloride, and HOE-694 with an inhibitory constant ( K i) of ∼480.2, 1.1, and 9.5 μM, respectively; 4) is inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, an anion exchange inhibitor at low concentration and a Cl channel blocker at high dose, and by 5-nitro-2(3-phenylpropylamino)benzoic acid, a Cl channel blocker, with a K i of ∼280.6 and 18.3 μM, respectively; and 5) substantially stimulated Cl-NHE activity by dietary Na depletion, which increases plasma aldosterone and inhibits NHE in surface cell AMV. These properties of Cl-NHE are distinct from those of NHE1, NHE2, and NHE3 isoforms that are present in colonic epithelial cells; thus these results suggest that the colonic crypt cell Cl-NHE is a novel NHE isoform.

Effect of pH on growth of mouse renal cortical tubule cells in primary culture

1989 ◽  
Vol 257 (3) ◽  
pp. C419-C426 ◽  
Author(s):  
S. S. Blumenthal ◽  
D. L. Lewand ◽  
M. A. Buday ◽  
N. S. Mandel ◽  
G. S. Mandel ◽  
...  
Keyword(s):  
Water Content ◽  
Prostaglandin E1 ◽  
Primary Cultures ◽  
Defined Medium ◽  
Thymidine Uptake ◽  
Medium Ph ◽  
Medium Acidification ◽  
Cell Ph ◽  
Glass Slides ◽  
Tubule Cells

We examined the effect of the medium pH on growth of primary cultures of mouse cortical tubule cells grown in defined medium. A significantly higher DNA content was observed within 24 h of lowering medium pH from 7.4 to 6.8 or 7.1 and persisted for the duration of the study. Further studies revealed that either medium acidification or insulin plus prostaglandin E1 nearly doubled uptake of [3H]thymidine in cells deprived of other growth factors for the previous 72-110 h. Moreover, the effects of insulin, prostaglandin E1, and medium acidification on [3H]thymidine uptake of quiescent cells were additive. An alkaline medium pH appeared to have a small but significant effect on cell hypertrophy, since cells exposed to pH 7.4 and 7.7 had a higher protein-to-DNA ratio than cells incubated at a lower pH. Cell pH of monolayers grown on glass slides determined from fluorescence of the carboxyfluorescein analogue 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) was linearly correlated with medium pH, and changes in medium pH resulted in changes in steady-state cell pH of a similar magnitude. Four hours after medium acidification, relative increases in cell Na+ and water content occurred, whereas medium alkalinization led to decreases in cell Na+ and water content. The increases in cell Na+ and cell water content at pH 6.8 could be inhibited by amiloride. We conclude that decreasing the cell pH can be a mitogenic stimulus for renal tubule cells. Medium acidification is accompanied by changes in cell Na+ transport, which may be mediated in part by altered Na+-H+ antiporter activity.

Sign in / Sign up

Export Citation Format

Share Document