Readdition Of Na Research Articles

Reversibility of (Ag,Cu)(In,Ga)Se2 electrical properties with the addition and removal of Na: Role of grain boundaries

The Na content of (Ag,Cu)(In,Ga)Se2 films was cyclically adjusted using a novel method involving cycles of water rinsing at 60 °C followed by heating in air at 200 °C to remove Na and evaporation of NaF to re-introduce Na back into the film. The low temperatures and short heating times ensure that Na is removed only from grain boundaries while leaving grain interiors unaffected. Cross-grain conductivity and Seebeck coefficient were measured during this removal procedure and both measurements decreased when Na was removed and both recovered upon the re-addition of Na, consistent with an increase in compensating donor defects in the absence of Na. These results demonstrate that Na reversibly affects the electrical properties of grain boundaries. We propose that Na reversibly passivates donor-like defects such as InCu double donors at grain boundaries.

Na+-dependent HCO3− Import by the slc4a10 Gene Product Involves Cl− Export

The slc4a10 gene encodes an electroneutral Na(+)-dependent HCO(3)(-) importer for which the precise mode of action remains unsettled. To resolve this issue, intracellular pH (pH(i)) recordings were performed upon acidification in the presence of CO(2)/HCO(3)(-) by 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) fluorometry of stably slc4a10-transfected NIH-3T3 fibroblasts. slc4a10 expression induced a significant Na(+)-dependent pH(i) recovery, which was accompanied by an increase in the intracellular Na(+) concentration evaluated by use of the Na(+)-sensitive fluorophore CoroNa Green. The estimated Na(+):HCO(3)(-) stoichiometry was 1:2. Cl(-) is most likely the counterion maintaining electroneutrality because (i) Na(+)-dependent pH(i) recovery was eliminated in Cl(-)-depleted cells; (ii) acute extracellular Cl(-) removal led to a larger alkalization in slc4a10-transfected cells than in control cells; and (iii) the 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS)-sensitive and Na(+)- and HCO(3)(-)-dependent (36)Cl(-)-efflux during pH(i) recovery was significantly greater in acidified slc4a10-transfected cells than in control cells. Charged amino acids specific to slc4a gene family members that transport Na(+) and are expected to move more HCO(3)(-) molecules/turnover were targeted by site-directed mutagenesis. Na(+)-dependent pH(i) recovery was reduced in each of the single amino acid mutated cell lines (E890A, E892A, H976L, and H980G) compared with wild type slc4a10-transfected cells and completely eliminated in quadruple mutant cells. In conclusion, the data suggest that slc4a10 expressed in mammalian cells encodes a Na(+)-dependent Cl(-)/HCO(3)(-) exchanger in which four specific charged amino acids seem necessary for ion transport.

Properties of the TRPML3 Channel Pore and Its Stable Expansion by the Varitint-Waddler-causing Mutation

TRPML3 is a H(+)-regulated Ca(2+) channel that shuttles between intracellular compartments and the plasma membrane. The A419P mutation causes the varitint-waddler phenotype as a result of gain-of-function (GOF). The mechanism by which A419P leads to GOF is not known. Here, we show that the TRPML3 pore is dynamic when conducting Ca(2+) to change its conductance and permeability, which appears to be mediated by trapping Ca(2+) within the pore. The pore properties can be restored by strong depolarization or by conducting Na(+) through the pore. The A419P mutation results in expanded channel pore with altered permeability that limits modulation of the pore by Ca(2+). This effect is specific for the A419P mutation and is not reproduced by other GOF mutations, including A419G, H283A, and proline mutations in the fifth transmembrane domain. These findings describe a novel mode of a transient receptor potential channel behavior and suggest that pore expansion by the A419P mutation may contribute to the varitint-waddler phenotype.

Intracellular pH regulation in isolated trout gill mitochondrion-rich (MR) cell subtypes: Evidence for Na +/H + activity

We have studied intracellular pH (pH i) recovery in isolated trout gill mitochondrion-rich (MR) cells following acidification by the NH 4Cl pre-pulse technique. Within a mixed MR cell population, one cell type displayed Na +-independent pH i recovery while the other cell type lacked a Na +-independent pH i recovery. Cells displaying Na + independent recovery exhibited a significantly higher buffering capacity compared to cells lacking Na +-independent pH i recovery. Cells displaying Na + independent recovery were identified as PNA + (peanut lectin agluttinin binding) MR cells while those unable to recover were identified as PNA − (non-peanut lectin agluttinin binding) MR cells. Therefore, recovery from acidification in the absence of Na + provides a direct functional marker for PNA + and PNA − MR cells. Re-addition of Na + to acidified cells resulted in a transient pH i recovery in both cell types. This event was abolished by amiloride (500 µM) but it was insensitive to phenamil (50 µM). The phorbol ester PMA (1 µM) potentiated the Na + induced pH i recovery suggesting that activation by PKC is required for continuous Na +/H + exchanger activity in trout gill MR cells. This study is the first functional description of pH i recovery in lectin-identified trout gill MR cells and provides insight into a putative cellular signaling mechanism that may control pH i regulation in the gill epithelium.

Sodium-dependent recovery of ionised magnesium concentration following magnesium load in rat heart myocytes

Our objectives were to investigate regulation of intracellular ionised Mg2+ concentration ([fMg2+]i) in cardiac muscle and cardiac Na+/Mg2+ antiport stoichiometry. [fMg2+]i was measured at 37 degrees C in isolated rat ventricular myocytes with mag-fura-2. Superfusion of myocytes with Na+ and Ca2+ free solutions containing 30 mM Mg2+ for 15 min more than doubled [fMg2+]i from its basal level (0.75 mM). Re-addition of Na+ caused [fMg2+]i to fall exponentially with time to basal level, the rate increasing linearly with [Na+]. Log(recovery rate) increased linearly with log([Na+]), the slope of 1.06 (95% confidence limits, 0.94-1.17) suggesting one Na+ ion is exchanged for each Mg2+. [fMg2+]i recovery was complete even if the membrane potential was depolarised to 0 mV or if superfusate [Mg2+] was increased to 3 mM. Recovery was rapid in normal Tyrode (0.3 min(-1)) with a Q10 of 2.2. It was completely inhibited by 200 microM imipramine but was unaffected by 20 microM KB-R7943 or 1 microM SEA0400, suggesting the Na+ /Ca2+ antiporter is not involved. Membrane depolarisation by increasing superfusate [K+] to 70 mM, or voltage clamp to 0 mV, increased recovery rate in Na+ containing solutions more than threefold. We conclude [fMg2+]i recovery is by Mg2+ efflux on a 1 Na+:1 Mg2+ antiport.

Differences in regulation of pH(i) in large (>/=10 nuclei) and small (

Osteoclasts are multinucleated cells that resorb bone by extrusion of protons and proteolytic enzymes. They display marked heterogeneity in cell size, shape, and resorptive activity. Because high resorptive activity in vivo is associated with an increase in the average size of osteoclasts in areas of greater resorption and because of the importance of proton extrusion in resorption, we investigated whether the activity of the bafilomycin A(1)-sensitive vacuolar-type H(+)-ATPase (V-ATPase) and amiloride-sensitive Na(+)/H(+) exchanger differed between large and small osteoclasts. Osteoclasts were obtained from newborn rabbit bones, cultured on glass coverslips, and loaded with the pH-sensitive indicator 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Intracellular pH (pH(i)) was recorded in single osteoclasts by monitoring fluorescence. Large (>/=10 nuclei) and small (</=5 nuclei) osteoclasts differed in that large osteoclasts had a higher basal pH(i), their pH(i) was decreased by bafilomycin A(1) addition or removal of extracellular Na(+), and the realkalinization upon readdition of Na(+) was bafilomycin A(1) sensitive. After acid loading, a subpopulation of large osteoclasts (40%) recovered by V-ATPase activity alone, whereas all small osteoclasts recovered by Na(+)/H(+) exchanger activity. Interestingly, in 60% of the large osteoclasts, pH(i) recovery was mediated by both the Na(+)/H(+) exchanger and V-ATPase activity. Our results show a striking difference between pH(i) regulatory mechanisms of large and small osteoclasts that we hypothesize may be associated with differences in the potential resorptive activity of these cells.

Mechanisms of basolateral Na+ transport in rabbit esophageal epithelial cells.

We examined the mechanisms of cellular Na+ transport, both Cl- dependent and Cl- independent, in the mammalian esophageal epithelium. Rabbit esophageal epithelium was dissected from its muscular layers and mounted in a modified Ussing chamber for impalement with ion-selective microelectrodes. In bicarbonate Ringer, transepithelial potential difference was -14.9 +/- 0.9 mV, the transepithelial resistance (RTE) was 1,879 +/- 142 Omega. cm2, the basolateral membrane potential difference (VmBL) was -53 +/- 1.5 mV, and the intracellular activity of Na+ (aNai) was 24.6 +/- 2.1 mM. Removal of Na+ and Cl- from the serosal and luminal baths decreased aNai to 6.6 +/- 0.6 mM. Readdition of Na+ to the serosal bath in the absence of Cl- increased aNai by 21.8 +/- 3.0 mM, whereas VmBL and RTE remained unchanged. When serosal Na+ was readded in the presence of amiloride the increase in aNai and the rate of Na+ entry were decreased by approximately 50%. 5-(N-ethyl-N-isopropyl)amiloride mimicked the effect of amiloride, whereas phenamil did not. Subsequent readdition of Cl- to the serosal bath further increased aNai by 4.4 +/- 1.9 mM. When the cells were acid loaded by pretreatment with NH+4 in nominally HCO-3-free Ringer, intracellular pH measurements showed a pHi recovery that is dependent on the presence of Na+ in the serosal bath and that can be blocked by amiloride. These data indicate that esophageal epithelial cells possess a Na+-dependent, amiloride-sensitive electroneutral mechanism for Na+ entry consistent with the presence of a basolateral Na+/H+ exchanger. The ability of Cl- to further enhance Na+ entry supports the existence of at least one additional Cl--dependent component of basolateral Na+ entry.

Evidence for a Na+/Ca2+ exchange mechanism in frog skin epithelium.

In the present study we investigated the possible existence of a Na+/Ca2+ exchange mechanism in the basolateral membrane of the frog skin epithelium and whether such a mechanism plays a role in the regulation of transepithelial Na+ transport. Cytosolic calcium ([Ca2+]i) was measured with the probe fura-2 in a set-up in which pieces of tissue were mounted on the stage of an epifluorescence microscope. Na+ transport was measured as the amiloride-sensitive short-circuit current (Isc) using a conventional voltage clamp. Basal [Ca2+]i was 65+/-6 nM (n=15). Removal of Na+ from the mucosal solution had no effect on [Ca2+]i. When Na+ was removed from the serosal solution, [Ca2+]i increased biphasically to a peak of 220+/-38 nM (n=8, P=0.006). Readdition of Na+ to the serosal solution returned [Ca2+]i to control level. The serosal Na+ gradient and changes in [Ca2+]i were closely correlated; stepwise changes in serosal Na+ were followed by stepwise changes in [Ca2+]i. These observations indicate the existence of a Na+/Ca2+ exchange mechanism in the basolateral membrane of the frog skin epithelium. The transepithelial Na+ transport decreased from 13.2+/-1.8 to 9.2+/-1.5 microA cm-2 (n=8, P=0.049) when Na+ was omitted from the serosal solution. When this protocol was repeated in the absence of serosal Ca2+, Na+ transport decreased similarly from 16.7+/-1.7 to 11.6 +/-1. 8 microA cm-2 (n=6, P=0.004). We conclude that it is unlikely that the observed decrease in Isc after removal of serosal Na+ is due to an increase in [Ca2+]i per se.

HCO 3−-dependent ion transport systems and intracellular pH regulation in colonocytes from the chick

The current study examines the presence of the Na +/HCO 3 − cotransporter and of the Cl −/HCO 3 − exchanger in chicken colonocytes and their role in cytosolic pH (pH i) homeostasis. pH i was measured with 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) at 25°C. Basal pH i was 7.16 in HEPES-buffered solutions and 7.06 in those buffered with HCO 3 −. Removal of external Cl − increased pH i and Cl − reinstatement brought the pH i towards resting values. These Cl −-induced pH i changes were Na +-independent, inhibited by H 2-DIDS and faster in the presence than in the absence of HCO 3 −. Cells recovered from alkaline loads by a mechanism that was Cl −-dependent, Na +-independent and inhibited by H 2-DIDS. This rate of Cl −-dependent cell acidification decreased as the pH i decreased, with a Hill coefficient value close to 4. Removal of external Na + decreased pH i and readdition of Na + brought pH i towards the control values. The rate of the Na +-induced changes was not modified by the presence of HCO 3 − and was prevented by EIPA and unaffected by H 2-DIDS. In the presence of EIPA cells partially recovered from a moderate acid load only when both Na + and HCO 3 − were present. The EIPA resistant Na +- and bicarbonate-dependent pH i recovery was inhibited by H 2-DIDS and occurred at equal rates in both Cl −-containing and Cl −-free solutions. It is concluded that in chicken colonocytes bathed in HCO 3 −-buffered solutions, both the Na +/H + exchanger and the Cl −/HCO 3 − exchanger participate in setting the resting pH i value. The latter transporter helps the cells to recover from alkaline loads and the first transporter, together with the Na +/HCO 3 − cotransporter, is involved in pH i recovery from an acid load.

Regulation of the renal Na-HCO3 cotransporter: VI. Mechanism of the stimulatory effect of protein kinase C

We have previously shown that the activity of the Na-HCO3 cotransporter is stimulated by protein kinase C (PKC) activation, but the mechanism responsible for this effect is not clear. We have shown that cultured proximal tubule cells of the rabbit have DIDS-sensitive Na-HCO3 cotransporter activity as assessed by HCO3-dependent 22Na uptake or by measurement of intracellular pH. In cells loaded with BCECF and treated with the amiloride analogue, ethylisopropyl amiloride, removal of extracellular Na was associated with a rapid decrease in pH which returned to normal with re-addition of Na. This pH recovery was inhibited by DIDS and was used to quantify the activity of the Na-HCO3 cotransporter. In the present study, we utilized primary cultures of the proximal tubule of the rabbit to examine the effect of PKC activation on the activity of the Na-HCO3 cotransporter. Short term incubation (5 min) with the active phorbol ester, phorbol 12-myristate, 13-acetate (PMA), 10(-7) M, caused a significant stimulation of the Na-HCO3 cotransporter activity as compared to controls. Incubation for two hours also caused a significant stimulation of the Na-HCO3 cotransporter activity. The inactive analogue of PMA, 4-alpha phorbol, failed to alter the cotransporter. Similar results were observed when we examined the effect of PMA on HCO3-dependent 22Na uptake. The effect of PMA to stimulate the cotransporter was mediated by PKC activation since it could be prevented by the PKC inhibitors, calphostin C or sphingosine, or by prior PKC depletion. The long term but not the short term effect of PMA to stimulate the Na-HCO3 cotransporter activity was prevented by the protein synthesis inhibitors, actinomycin D or cycloheximide. The early effect of PKC to stimulate the cotransporter appeared to be associated with increased phosphorylation of a 56 kD protein band, while the late effect appeared to be associated with an increase in immunoreactive content of a 56 kD protein which is thought to be an active component of the cotransporter. Thus PKC stimulation activates the Na-HCO3 cotransporter by two distinct mechanisms: a long term effect which is protein synthesis-dependent and a short term effect which is protein synthesis-independent and is likely mediated by phosphorylation.

Na+-HCO3(-) cotransporter and intracellular pH regulation in chicken enterocytes.

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

Regulation of renal Na-HCO3 cotransporter: III. Presence and modulation by glucocorticoids in primary cultures of the proximal tubule

We looked for the presence of the Na-HCO3 cotransporter in primary cultures of the proximal tubule and examined the modulation of this cotransporter by glucocorticoid hormones. Primary cultures of the proximal tubule of the rabbit have Cl-independent, HCO3-dependent 22Na uptake which is DIDS-sensitive. In addition, in cells loaded with BCECF and perfused with Cl-free solution, removal of Na was associated with a decrease in intracellular pH which returned to normal with re-addition of Na. The pH recovery was not inhibited by EIPA but was sensitive to DIDS. These findings are compatible with existence of Na-HCO3 cotransporter in these cells. We examined the role of glucocorticoids on the activity of the Na-HCO3 cotransporter by culturing proximal tubule cells in the presence of hydrocortisone and when confluence was reached, hydrocortisone was deleted from the medium. In the absence of hydrocortisone, the activity of the cotransporter, measured either isotopically or fluorometrically, was significantly decreased, whereas re-addition of hydrocortisone 10(-8) M, restored the activity of the cotransporter to normal levels. The effect of hydrocortisone could not be duplicated by aldosterone, suggesting a glucocorticoid-dependent effect. Dexamethasone, a glucocorticoid without mineralocorticoid activity, stimulated the activity of the cotransporter within physiologic concentrations and this effect was blocked by progesterone. The effect of dexamethasone was time-dependent and was prevented by cycloheximide, a protein synthesis inhibitor. These results demonstrate that primary cultures of the proximal tubule have Na-HCO3 cotransporter activity which is modulated by physiological concentrations of glucocorticoids through a protein synthesis-dependent mechanism.

Sodium-bicarbonate cotransport current in identified leech glial cells.

1. The membrane current associated with the cotransport of Na+ and HCO3- was investigated in neuropil glial cells in isolated ganglia of the leech Hirudo medicinalis L. using the two-electrode voltage-clamp technique. 2. The addition of 5% CO2-24 mM HCO3- evoked an outward current, which slowly decayed, and which was dependent upon the presence of external Na+. Removal of CO2-HCO3- elicited a transient inward current. Re-addition of Na+ to Na(+)-free saline in the presence of CO2-HCO3- also produced an outward current. Under these conditions an intracellular alkalinization and a rise in intracellular [Na+] were recorded using triple-barrelled, ion-sensitive microelectrodes. Addition or removal of HCO3-, in the absence of external Na+, caused little or no change in membrane voltage, membrane current and intracellular pH, indicating that the glial membrane has a very low HCO3- conductance. 3. Voltage steps revealed nearly linear current-voltage relationships both in the absence and presence of CO2-HCO3-, with an intersection at the assumed reversal potential of the HCO(3-)-dependent current. These results suggest a cotransport stoichiometry of 2HCO3-: 1 Na+. The HCO(3-)-dependent current could be inhibited by diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). 4. Simultaneous recording of current and intracellular pH showed a correlation of the maximal acid-base flux with the transient HCO(3-)-dependent current during voltage steps in the presence of CO2-HCO3-. The maximum rate of acid-base flux and the HCO(3-)-dependent peak current showed a similar dependence on membrane voltage. Lowering the external pH from 7.4 to 7.0 produced an inward current, which increased twofold in the presence of CO2-HCO3-. This current was largely inhibited by DIDS, indicating outward-going electrogenic Na(+)-HCO3- cotransport during external acidification. 5. When external Na+ was replaced by Li+, a similar outward current and intracellular alkalinization were observed in the presence of CO2-HCO3-. The Li(+)-induced intracellular alkalinization was not inhibited by amiloride, a blocker of Na+(Li+)-H+ exchange, but was sensitive to DIDS. These results suggest that Li+ could, at least partly, substitute for Na+ at the cotransporter site. 6. Our results indicate that the Na(+)-HCO3- cotransport produces a current across the glial cell membrane in both directions with a reversal potential near the membrane resting potential, rendering pHi a function of the glial membrane potential.

Calcium fluxes in rat thyroid FRTL-5 cells. Evidence for a functional Na+/Ca2+exchange mechanism

The effect of extracellular Na+ on cytosolic free Ca2+ and on influx and efflux of Ca2+ was investigated in FRTL-5 thyroid cells. Stimulating the cells with the purinergic agonist ATP induced a rapid efflux of 45Ca2+ from cells loaded with 45Ca2+. Replacement of extracellular Na+ with choline+, significantly decreased the adenosine triphosphate-induced efflux of 45Ca2+. Furthermore, adenosine triphosphate-induced uptake of 45Ca2+ was increased when extracellular Na+ was replaced with choline+, compared with the uptake seen in Na+ buffer. Replacing extracellular Na+ with choline+, increased resting levels of cytosolic free Ca2+ from 50 +/- 2 nM (mean +/- SE) to 81 +/- 3 nM (P less than 0.05) in Fura 2 loaded cells. In cells preincubated with 1 mM ouabain for 30 min, resting cytosolic free Ca2+ increased to 73 +/- 3 nM (P less than 0.05). In a Na+ buffer, the adenosine triphosphate-induced transient increase in cytosolic free Ca2+ was 872 +/- 59 nM, compared with 1070 +/- 63 nM in choline+ buffer (P less than 0.05). The plateau level of cytosolic free Ca2+ in response to adenosine triphosphate was 130 +/- 16 nM in Na+ buffer, compared with 209 +/- 9 nM in choline+ buffer (P less than 0.05). Readdition of Na+ to the plateau phase decreased cytosolic free Ca2+ to 152 +/- 5 nM. Stimulating the cells with 10 microM of the Na(+)-selective monovalent ionophore monensin increased cytosolic free Ca2+ from 53 +/- 9 nM to 124 +/- 16 nM (P less than 0.05). This increase in cytosolic free Ca2+ was dependent on both extracellular Na+ and extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

H+ extrusion by an apical vacuolar-type H(+)-ATPase in rat renal proximal tubules.

The activity of Na+/H(+)-exchange and H(+)-ATPase was measured in the absence of CO2/HCO3 by microfluorometry at the single cell level in rat proximal tubules (superficial S1/S2 segments) loaded with BCECF [2'7'-bis(carboxyethyl)5-6-carboxyfluorescein- acetoxymethylester]. Intracellular pH (pHi) was lowered by a NH4Cl-prepulse technique. In the absence of Na+ in the superfusion solutions, pHi recovered from the acid load by a mechanism inhibited by 0.1 microM bafilomycin A1, a specific inhibitor of a vacuolar-type H(+)-ATPase. Readdition of Na+ in the presence of bafilomycin A1 produced an immediate recovery of pHi by a mechanism sensitive to the addition of 10 microM EIPA (ethylisopropylamiloride), a specific inhibitor of Na+/H+ exchange. The transport rate of the H(+)-ATPase is about 40% of Na+/H(+)-exchange activity at a similar pHi (0.218 +/- 0.028 vs. 0.507 +/- 0.056 pH unit/min. Pre-exposure of the tubules to 30 mM fructose, 0.5 mM iodoacetate and 1 mM KCN (to deplete intracellular ATP) prevented a pHi recovery in Na(+)-free media; readdition of Na+ led to an immediate pHi recovery. Tubules pre-exposed to Cl(-)-free media for 2 hr also reduced the rate of Na(+)-independent pHi recovery. In free-flow electrophoretic separations of brush border membranes and basolateral membranes, a bafilomycin A1-sensitive ATPase activity was found to be associated with the brush border membrane fraction; half maximal inhibition is at 6 x 10(-10) M bafilomycin A1.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of Na+ replacement on intracellular pH and [Ca2+] in rabbit salivary gland acinar cells.

1. The role of Na(+)-dependent mechanisms in regulating the intracellular pH (pHi) and free calcium concentration ([Ca2+]i) in acinar cells of the rabbit mandibular salivary gland was examined. The fluorescent dyes BCECF and Fura-2 were used to measure pHi and [Ca2+]i respectively in suspensions of isolated acini. 2. Replacement of all the extracellular Na+ with N-methyl-D-glucamine (NMDG) decreased resting pHi from a control value of 7.1-7.2 to 6.8-6.9. Re-addition of Na+ or Li+ caused a recovery of pHi towards control values. This recovery was blocked by 10-50 microM-ethylisopropylamiloride (EIPA), suggesting that it was mediated by Na(+)-H+ exchange. The rate of recovery of pHi when Na+ was re-introduced increased with Na+ concentration with an apparent Km for Na+ of around 30 mM. 3. Replacement of all of the extracellular Na+ with Li+ caused only a small decrease in resting pHi. 4. Stimulation of acini with 1 microM-acetylcholine (ACh) evoked an intracellular acidosis both under control conditions and when acini were bathed in Na(+)-free media. Following the acidosis pHi recovered in acini bathed in either control medium or Na(+)-free (Li+) medium, but not in acini bathed in Na(+)-free (NMDG) medium or in control medium containing EIPA. 5. Stimulation of acini bathed in Na(+)-free, HCO(3-)-free medium with ACh did not cause any change in pHi. 6. Re-addition of Na+ to acini bathed in Na(+)-free, HCO(3-)-free medium evoked the same rate of alkalinization whether or not the acini had been stimulated with ACh, suggesting that receptor stimulation per se did not lead to an activation of acid extrusion. 7. Resting [Ca2+]i was elevated in acini bathed in Na(+)-free (NMDG) medium, but not in acini bathed in Na(+)-free (Li+) medium. 8. ACh evoked a maintained rise in [Ca2+]i in acini bathed in control medium and in Na(+)-free media with either NMDG or Li+ as the Na+ substitute. 9. Experiments in which external Ca2+ was reduced to low levels (by the addition of EGTA) just prior to addition of ACh showed that ACh released intracellular Ca2+ stores under both control and Na(+)-free conditions. 10. In acini bathed in Na(+)-free (NMDG) solution and stimulated with ACh, re-addition of either Na+ or Li+ reduced [Ca2+]i. The reduction of [Ca2+]i on Na+ re-addition was blocked by EIPA. [Ca2+]i could also be reduced under these conditions by alkalinizing the cytosol using the weak base trimethylamine.(ABSTRACT TRUNCATED AT 400 WORDS)

Extracellular Na+ dependency of free cytosolic Ca2+ regulation in aortic vascular smooth muscle cells

This study examined contribution of Na(+)-dependent processes to the regulation of free cytosolic calcium (Ca2+i) in cultured vascular smooth muscle cells (VSMC) using fura-2. Removal of Na+ from superfusate (replacement with choline) resulted in an increment of Ca2+i that was greatly augmented by pretreatment with ouabain. Under both conditions, Ca2+i increase was followed by partial recovery to a new steady state that was still significantly higher than that seen before removal of external Na+ (Na+o). In ouabain-pretreated cells lowering of Na+o caused progressive increases in Ca2+i. Addition of NiCl2, a Na(+)-Ca2+ exchange inhibitor, completely blocked the increase in Ca2+i produced by removal of Na+o, indicating that the Na(+)-Ca2+ antiporter was responsible for observed Ca2+i changes. Ca2+i increase produced by reduction of Na+o was also seen after depletion of inositol trisphosphate-sensitive Ca2+ stores with repeated pulses of angiotensin II or after blockade of sarcoplasmatic reticulum Ca2+ release with TMB-8 but was not observed in the absence of external Ca2+. These observations indicate that the source of Ca2+i increase in response to changes in the transmembrane Na+ gradient is largely external, and potentiation of the Ca2+i surge by ouabain suggests Ca2+ influx via the Na(+)-Ca2+ exchanger operating in the reverse mode. The relative contribution of a Na(+)-dependent and -independent component of Ca2+i recovery was investigated by superfusing cells with ionomycin in a Na(+)-free medium and later adding Na+ to the medium. This Ca2+ ionophore increased Ca2+i to a peak, and this was followed by a rapid but partial recovery to a new steady state. Readdition of varying amounts of Na+ to the superfusate, in the continued presence of ionomycin, resulted in concentration-related decline in Ca2+i, thereby uncovering a substantial contribution of a Na(+)-dependent mechanism of Ca2+i regulation. Decline of Ca2+i produced by readdition of Na+ was blocked by addition of NiCl2 to the superfusate. Our findings thereby provide evidence for Ca2+i regulation in VSMC via a Na(+)-dependent mechanism, consistent with a Na(+)-Ca2+ exchanger, which acts as a Ca2+ efflux mechanism when Ca2+i is elevated. Na(+)-Ca2+ exchanger acts as a Ca2+ influx mechanism when intracellular Na+ is elevated by prior exposure to ouabain.

Identification and characteristics of a Na+/Ca2+ exchanger in cultured human mesangial cells

Excitable cells express Na+/Ca2+ exchange activity among other mechanisms modulating rapid fluctuations of cytosolic free Ca2+ ([Ca2+]i). We studied functions and regulation of a Na+/Ca2+ exchanger in cultured human glomerular mesangial cells. Fura-2-loaded confluent monolayers reacted to removal of ambient Na+ with an immediate, transient elevation of [Ca2+]i, assessed by single/dual wavelength fluorometry. Peak [Ca2+]i was inversely correlated with the extracellular Na+ concentration. Ca2+ influx was the sole mechanism implicated, as the [Ca2+]i rise was prevented by EGTA. The process was inhibited by 1 mM amiloride, but not by blockers of voltage-operated Ca2+ channels. Re-addition of Na+ resulted in a rapid decrease of [Ca2+]i, indicating bimodal operation of the exchanger. Na(+)-loading the cells with monensin and ouabain enhanced Ca2+ uptake. Prior stimulation of [Ca2+]i with the thromboxane A2 mimetic, U-46619, or angiotensin II also increased Ca2+ uptake upon subsequent Na+ removal, suggesting induction of the exchanger by vasoconstrictors. Moreover, the magnitude of agonist-induced [Ca2+]i transients was amplified by Na+ removal, indicating that the exchanger modulates the effects of vasoconstrictors. These results demonstrate that an inducible Na+/Ca2+ antiporter is operative in resting and stimulated human mesangial cells, further confirming their smooth muscle origin and potential regulatory role on glomerular hemodynamics.

PH regulatory transport systems in a smooth muscle-like cell line

The membrane transport systems responsible for pH regulation in BC3H-1 cells were studied using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). In nominally CO2-free Na N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (NHB) recovery from acidification after an NH4Cl pulse was reversibly inhibited by 1 mM amiloride or by Na-free solutions. On exposure to 5% CO2-HCO3 (external pH constant at 7.4), BC3H-1 cells alkalinized by approximately 0.3-0.4 pH unit. This CO2-induced alkalinization was unaffected by 1 mM amiloride, markedly reduced by 0.5 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), and inhibited by Na-free solutions. On readdition of Na, cells rapidly alkalinized, even in the presence of 1 mM amiloride. Exposure to Cl-free CO2-HCO3 solutions caused a rapid alkalinization of nearly 1 pH unit that was abolished by SITS, largely independent of Na, unaffected by amiloride, and unchanged by membrane depolarization in high external K solutions. CO2-induced alkalinization was slowed by approximately 75% after prolonged exposure of cells to Cl-free NHB, but a distinct recovery from acidification remained in these Cl-depleted cells. This recovery was Na-dependent, SITS-inhibitable, and unaffected by depolarization in high-K solutions. In the presence of CO2, the acidification seen in response to NH4Cl-induced alkalinization was reduced 50% by 0.5 mM SITS. These data suggest that the regulation of pH in BC3H-1 cells is mediated by at least three transport systems: 1) Na-H exchange; 2) Cl-HCO3 exchange; and 3) electroneutral (Na + HCO3)-Cl exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Na+ and K+ on Cl‐ distribution in guinea‐pig vas deferens smooth muscle: evidence for Na+, K+, Cl‐ co‐transport.

1. Smooth muscle cells of the guinea-pig vas deferens after Cl- depletion actively reaccumulate ions to a level many times higher than that predicted by a passive distribution, even when anion exchange (largely responsible for Cl- movements in this preparation) is inhibited by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid). The cells therefore must possess a second mechanism for Cl- accumulation. We have now investigated the ionic requirement of this mechanism using a combination of ion analysis, 36Cl fluxes and direct measurement of the intracellular Cl- activity (aiCl). 2. In the steady state, the Cl- content of tissues was 12-16% less in Na(+)-free solution than in normal Krebs solution. 3. Loss of 36Cl into Cl(-)-free solution was slowed by the absence of Na+ and accelerated on its readdition. Uptake of 36Cl by Cl(-)-depleted tissues was also reduced in the absence of extracellular Na+, particularly at longer time intervals as uptake reached completion. These effects occurred in the presence and absence of CO2-HCO3- and in the presence of DIDS. 4. The initial rate of rise of aiCl on readdition of Cl- to Cl(-)-depleted cells was not significantly affected by the absence of Na+ in the presence of a functional anion exchange, but aiCl stabilized at a lower value than in normal solution. Readdition of Na+ stimulated a rise in aiCl to the control level. Removal and readdition of K+ under these conditions had negligible effects. 5. When anion exchange was inhibited by the presence of DIDS, removal and readdition of Na+ caused, respectively, a marked inhibition and stimulation of the rise in aiCl during Cl- reaccumulation. Under these conditions removal and readdition of K+ had similar effects. 6. The results suggest that Na+, K+, Cl- co-transport is involved in transmembrane movements of Cl- at least when the anion exchange mechanism is blocked. 7. The possibility that the marked effects of changes in external Na+ on transmembrane Cl- movements in the presence of a functional anion exchange mechanism are caused by secondary effects due to changes in intracellular pH as well as to suppression of Na+, K+, Cl- co-transport is discussed.