Removal Of Na Research Articles

A Microplate Assay Measuring Chloride Ion Channel Activity

A microplate chloride ion channel assay, usingN-(6-methoxyquinolyl) acetoethyl ester (MQAE) fluorescence changes has been developed. Forskolin stimulation of T84 cells caused cAMP-dependent, increased Cl−loss. Forskolin responses after 6 min gave an EC50of 0.27 ± 0.05 μm, illustrating the reproducibility of the assay. Forskolin exposure of CFPAC-1 cells, containing ΔF508 CFTR, and CFPAC-1 PLJ4.7 cells, transfected with WT-CFTR stimulated Cl−secretion only from the latter, showing that the assay can be used to measure CFTR function. Stimulation of CFPAC-1 cells with ionomycin increased Cl−efflux, demonstrating functional Ca2+-mediated Cl−secretion in these cells. Ionomycin also induced a dose-responsive Cl−efflux from T84 cells that, unlike the forskolin response, was Ca2+dependent. Removal of Na+ions severely inhibited basal and stimulated Cl−efflux from T84 cells. However, furosemide did not affect forskolin-stimulatedJCl, although the magnitude of the Cl−loss was reduced. The Stern–Volmer constant for MQAE fluorescence in T84 cells was calculated as 28.3 ± 0.9m−1and the [Cl−]iin untreated T84 cells was determined as 52.4 ± 0.6 mm. Stimulation of T84 cells with ionomycin and forskolin before inducing Cl−efflux allowed calculation of initial efflux rates without interference by second messenger generation and ion channel activation kinetics.

Hyperosmolality stimulates Na-K-ATPase gene expression in inner medullary collecting duct cells.

Primary cultures of inner medullary collecting duct (IMCD) cells of rats were incubated in hyperosmotic media to determine the effects on Na-K-ATPase alpha 1- and beta 1-subunit mRNA expression. Osmolality of the incubation media was raised from 300 up to 500 mosmol/kgH2O by adding NaCl, mannitol, raffinose, or urea. Hyperosmotic media supplemented with NaCl, mannitol, or raffinose caused two- to fourfold increases in the alpha 1-subunit mRNA accumulation and five- to eightfold increases in the beta 1-subunit mRNA accumulation, with peak elevations of both subunits at 12 h after addition. In sharp contrast, hyperosmolar urea medium had no effect at any time. When NaCl or mannitol was added to the media in amounts ranging from 300 to 600 mosmol/kgH2O, the maximal effects on both alpha 1- and beta 1-subunit mRNA accumulation occurred at 500 mosmol/kgH2O. In urea-supplemented medium, however, there was no significant change at any level of osmolality. The upregulation of alpha 1- and beta 1-subunit mRNA induced by hyperosmotic mannitol- or raffinose-supplemented media was markedly inhibited by removal of Na from the culture medium. Furthermore, pretreatment with a protein synthesis inhibitor cycloheximide partially inhibited the upregulation of alpha 1- and beta 1-subunit mRNA in IMCD cells exposed to hyperosmotic media treated with NaCl or mannitol. When IMCD cells were incubated with hyperosmotic media (500 mosmol/kgH2O) supplemented with NaCl or mannitol for 24 h, Na-K-ATPase activity increased by 78.6 and 82.8%, respectively. In contrast, hyperosmolar urea medium had no significant effect on Na-K-ATPase activity. These results demonstrate that 1) hyperosmolality induced by the poorly permeating solutes (NaCl, mannitol, and raffinose) but not the rapidly permeating solute (urea) stimulates both alpha 1- and beta 1-subunit mRNA accumulations in IMCD cells in a time- and an osmolality-dependent manner, 2) the hyperosmolality-induced upregulation of alpha 1- and beta 1-subunit mRNA leads to an increase in Na- K -ATPase activity; and 3) the above upregulation of alpha1- and beta 1-subunit mRNA in response to hyperosmotic media requires, at least in part, the presence of Na in the extracellular medium and the de novo synthesis of intermediate proteins.

The metamorphosed molybdenum vein-type deposit of the Alpeinerscharte, Tyrol (Austria) and its relation to Variscan granitoids

The molybdenite deposit of the Alpeinerscharte (Austria) is situated in Variscan greenschist- to amphibolite-facies metamorphosed granodiorites and granites of the western Tauern Window. These granitoids represent strongly fractionated calc-alkaline I-type magmas with minor S-type components and reveal post-orogenic affinities. Molybdenum contents (average 4.3 ppm) are slightly above the general background of average granites. Molybdenite mineralization is restricted to narrow quartz veins and quartz vein selvages which are presently composed of biotite and (almandine-grossular) garnet. These selvages show geochemical features typical of intermediate argillic alteration in a hydrothermal system postdating granite intrusion: instability of plagioclase causes removal of Na, Ba, Sr, Pb and Eu, while K and Ca remain nearly constant. Rare earth elements (apart from Eu) and metals are extremely enriched. Application of Fe-Mg exchange (garnet-biotite) and oxygen isotope (quartz-garnet, quartz-plagioclase) geothermometers to vein selvage mineral assemblages reveals temperatures of the late-Alpine (35–55 Ma) metamorphic overprint (∼540°C, 7–10 kbar). Leucocratic rocks composed of mainly orthoclase and plagioclase are occasionally spatially related to molybdenite-bearing veins; they are interpreted as episyenites formed by hydrothermal alteration of the host granite. This episyenitic alteration is characterized by a mass loss of ca. 30%, relative enrichment of plagioclase components, extreme depletion of Si, and minor depletion of Fe, Zn, Cu and Mo.

Differential regulation of Na+-K+-ATPase gene expression by corticosteriods in vascular smooth muscle cells.

To determine whether gluco- and mineralocorticoids have specific actions on Na+-K+-ATPase gene expression in vascular tissue, we used Northern blot analysis to compare the effects of dexamethasone (Dex) and aldosterone (Aldo) on Na+-K+-ATPase alpha1 and beta1-subunit mRNA expression in cultured vascular smooth muscle cells from rat aortae. Dex at 10(-6)M increased alpha1 -mRNA level 2.5-fold at 24 h and beta1-mRNA level 9.9-fold at 12 h. Aldo at 10(-6)M increased alpha1-mRNA 2.7-fold at 48 h and beta1-mRNA level 10.9-fold at 6 h. The half-maximal stimulation of both alpha1 and beta1-mRNA levels occurred at a concentration of 5-7 X 10(-9)M Dex, whereas it occurred at a concentration of 2-3 X 10(-9)M Aldo. The glucocorticoid receptor antagonist RU-38486 inhibited both Dex- and Aldo-mediated induction of beta1-mRNA. The mineralocorticoid receptor antagonist spironolactone inhibited Aldo-mediated induction of beta1-mRNA, whereas it had no effect on Dex-mediated induction of beta1-mRNA. Removal of Na+ from the extracellular medium (isosmotic replacement with choline) caused no effect on Dex-mediated induction beta1-mRNA, whereas it inhibited Aldo-mediated induction of beta1-mRNA. Addition of a specific inhibitor of the Na+/H+ exchange, ethylisopropylamiloride, had no effect on Dex-mediated induction of beta1-mRNA, whereas it resulted in a significant inhibition of Aldo-mediated induction of beta1-mRNA. We conclude that 1) both Dex and Aldo induce Na+-K+-ATPase alpha1- and beta1-mRNA expression in a time- and dose-dependent manner; 2) Dex-mediated induction of beta1-mRNA occurs only through glucocorticoid receptors, whereas Aldo-mediated induction of beta1-mRNA occurs through both gluco- and mineralocorticoid receptors; and 3) Dex-mediated induction of beta1-mRNA occurs through Na+-independent mechanisms, whereas Aldo-mediated induction of beta1-mRNA, at least in part, occurs through Na+-dependent mechanisms, including stimulation of the Na+/H+ exchange.

Acid-base transport in isolated rabbit duodenal villus and crypt cells.

Duodenal mucosal bicarbonate secretion is an important first line of defense against gastric acid. Studies in the ileum indicate that the secretion originates from the crypt cells, whereas villus cells are mainly absorptive. Data on acid/base transporters along the crypt-villus axis in duodenal epithelia are not available. It was our purpose to identify and compare acid/base transporters in isolated mammalian duodenal villus and crypt cells. The proximal duodenum of rabbits was excised, and duodenal epithelial cells were isolated in five fractions by a modified calcium chelation technique. Intracellular pH (pHi) was measured with a pH-sensitive dye and dynamic fluorescence ratio imaging. In both villus and crypt cells incubated in Hepes buffer, removal of Na+ or addition of amiloride decreased basal pHi and pHi recovery after intracellular acidification, indicating an Na+/H+ exchanger in both cell types. In both cell types acid extrusion rates in bicarbonate-buffered Ringer's solution were significantly higher than in Hepes buffer. The bicarbonate-dependent acid extruder was unaffected by removal of Cl- or addition of amiloride but was blocked by removal of Na+, indicating the presence of a NaHCO3 cotransporter in both villus and crypt cells. Removal of external Cl induced a reversible increase in pHi (inhibited by H2DIDS) in both villus and crypt cells, indicating a Cl-/HCO3- exchanger in both. Mammalian duodenal villus and crypt cells have identical acid-base transporters. These findings tend to negate the theory of a functional difference in acid-base transporters between duodenal villus and crypt cells and instead imply alkaline secretion by both cell fractions. However, as these experiments were performed in unpolarized, single cells, additional studies with either membrane vesicles or polarized cells are needed.

Effects of Ca2+ on rabbit translens short-circuit current: evidence for a Ca2+ inhibitable K+ conductance.

To characterize the effects of medium Ca2+ levels on rabbit lens electrical properties. Early studies with wholly submerged lenses had shown that Ca2+ removal from the bath resulted in an increased Rb+ efflux, a consequence of an increased Na+ Permeability and lens depolarization. Lenses were bathed with Ussing-type chambers under short-circuited conditions, an arrangement in which the translens short-circuit current (Isc) is carried out across the posterior lens surface mainly by an influx of Na+, and across the anterior face largely by a K+ efflux. Under the present conditions in which the effects of Ca2+ were characterized unilaterally, the above established effects could only be ascribed to the posterior surface. When Ca2+ removal was limited to the anterior face, the Isc increased from 11.87 +/- 1.17 to 17.04 +/- 1.52 microA/cm2 (means +/- SE's, n = 18; an accompanying translens resistance (Rt) decrease of 0.23 +/- 0.049 K omega.cm2 was also recorded). Conversely, increasing the control, anterior-bath [Ca2+] from 1.8 to 3.6 mM reduced the K+ efflux-dependent Isc from 10.54 +/- 1.09 to 8.93 +/- 1.02 (n = 10, with an Rt increase of 0.11 +/- 0.013). These changes were reversible Na(+)-independent, and fully inhibited by the presence of K+ channel blockers (quinidine or Ba2+). Inhibitions of the Ca2+ effects were also obtained with strontium, a Ca2+ surrogate. The Isc was less responsive to changes in the Ca2+ content of the posterior bath. Removal of the cation caused a gradual 1.65 +/- 0.72 microA/cm2 increase (n = 9, with an Rt decrease of 0.090 +/- 0.021 K omega.cm2). In the absence of posterior Na+, Ca2+ withdrawal resulted in highly variable responses, with some specimens exhibiting salient current increases, suggesting that an outwardly directed, posterior efflux of an anion could also have been affected. During the course of this study it was consistently observed that the removal of Na+ from the anterior bath led to an Isc decrease of 2.62 +/- 0.22 microA/cm2 (n = 32, with an Rt increase of 0.35 +/- 0.029 k omega.cm2). This change occurred in both the presence of ouabain and the absence of Ca2+, suggesting that it did not result from an inhibition of the Na(+)-K+ pump current nor from a reversal in putative Na+/Ca2+ exchange activity. Small Isc increases upon anterior Na+ withdrawal (1.68 +/- 0.17, n = 7), consistent with Na+ efflux from the lens, could only be observed with K+ channels inhibited with Ba2+. Also congruent with the observations of a relatively limited anterior Na+ permeability, was the finding that the induction of nonspecific cation channels with amphotericin B reduced the Isc by following Na+ from the anterior bath to enter the lens. Thus, changes in lens Isc can differentiate changes in K+ permeability across the native anterior epithelium from changes in Na+ permeability. Overall, these results suggest that lens Ca2(+)-mobilizing agents (e.g. acetylcholine) could trigger the inhibition of epithelial K+ conductance(s) by the direct action of Ca2+ on K+ channels.

Nifedipine and tetrodotoxin delay the onset of methylmercury-induced increase in [Ca2+]i in NG108-15 cells.

Methylmercury (MeHg) causes a multiphasic disruption of intraneuronal cation regulation. Release of Ca2+ from internal stores and entry of extracellular Ca2+ (Ca2+e) contribute to the temporally distinct early (first Ca2+ phase) and late (second Ca2+ phase) components of increased intracellular Ca2+ concentration ([Ca2+]i). The present study was designed to explore the mechanisms mediating the second Ca2+ phase. Fluorescence intensity was monitored from single NG108-15 cells loaded with fura-2 before and during acute application of 2 microM MeHg. Nifedipine (1 or 10 microM but not 0.1 microM) significantly delayed the time-to-onset of the second Ca2+ phase. Nifedipine (1 microM but not 0.1 microM) also caused a concentration-dependent delay in the onset of both the first Ca2+ phase which is independent of Ca2+e and the elevation of non-Ca2+ cation (non-Ca2+ phase). The L-type dihydropyridine (DHP) Ca2+ channel agonist Bay K-8644 (10 nM) had no effect on the time-to-onset of the second Ca2+ phase. Neither the N-type Ca2+ channel blocker omega-conotoxin GVIA (up to 1 microM) nor the nonselective Ca2+ channel blocker Ni2+ (1 mM) altered the time-to-onset of the second Ca2+ phase. Removal of Na+e or addition of the voltage-dependent Na+ channel antagonist tetrodotoxin (TTX, 1 microM) significantly delayed the onset of the second Ca2+ phase. In a manner similar to that for 1 microM nifedipine, TTX also delayed the onset of the other phases. Thus, we hypothesize that MeHg depolarizes the plasma membrane leading to an increase in the activation of voltage-dependent Na+ and Ca2+ channels which promotes, directly or indirectly, the influx of Ca2+ during the second Ca2+ phase.

Activation of nicotinic acetylcholine receptors expressed in quail fibroblasts: effects on intracellular calcium.

1. The aim of these experiments was to determine the ability of the muscle-type nicotinic acetylcholine receptor (AChR) stably expressed in quail fibroblasts (QF18 cells) to elevate intracellular calcium ([Ca2+]i) upon activation. Ratiometric confocal microscopy, with the calcium-sensitive fluorescent dye Indo-1 was used. 2. Application of the nicotine agonist, suberyldicholine (SDC), to the transfected QF18 cells caused an increase in [Ca2+]i. Control [Ca2+]i levels in QF18 cells were found to be 164 +/- 22 nM (mean +/- s.e. mean; n = 40 cells) rising to 600 +/- 81 nM on addition of SDC (10 microM; n = 15 cells), whereas no increase in [Ca2+]i was seen in non-transfected control QT6 fibroblasts (before: 128 +/- 9 nM, n = 40; after; 113 +/- 13 nM, n = 15). 3. The increase in [Ca2+]i caused by application of SDC was dose-dependent, with an EC50 value of 12.7 +/- 5.9 microM (n = 14). 4. The responses to SDC in QF18 cells were blocked by prior application of alpha-bungarotoxin (200 nM), by the addition of Ca2+ (100 microM), by removal of Na+ ions from the extracellular solution, or by the voltage-sensitive calcium channel blockers nifedipine and omega-conotoxin, which act with IC50 values of 100 nM and 100 pM respectively. 5. We conclude that activation of the nicotinic AChRs leads to a Na(+)-dependent depolarization and hence activation of endogenous voltage-sensitive Ca2+ channels in the plasma membrane and an increase in [Ca2+]i. There is no significant entry of Ca2+ through the nicotinic receptor itself.

Amiloride-sensitive Na + conductance in native Xenopus oocytes

Endogenous Na + conductances in the plasma membrane of oocytes of the South African clawed toad Xenopus laevis were investigated by microelectrode techniques and influx measurements. Removal of Na + from the bath solution under voltage clamp conditions led to a decrease in the clamp current indicating the existence of native Na + conductances. The observed current was voltage dependent but showed no marked rectification. Amiloride (10 μM) blocked this Na + current reversibly. However, amiloride analogues such as benzamil and phenamil had no effect on this Na + conductance. The Na +/H +-exchanger blocker EIPA (5-( N-ethyl- N-isopropyl)amiloride), another amiloride analogue, also had no effect thereby excluding a possible involvement of the Na +/H + exchanger. The Na + mediated current had a reversal potential of about 50 mV suggesting high selectivity of these Na + conductances for Na + over other monovalent cations. When Na + was replaced by K + in the bath solution, amiloride had no effect on the clamp current over the whole potential range demonstrating that only Na + but not K + can enter the cell via the investigated conductances. In radio tracer experiments 22Na + influx into oocytes was nearly halved in presence of amiloride (10 μM), whereas benzamil and phenamil again failed to influence 22Na + influx. These results suggest that the endogenous amiloride-sensitive Na + conductance belongs to a new class of channels which is quite different from amiloride-sensitive epithelial Na + channels.

Ca2+ influx induced by store release and cytosolic Ca2+ chelation in Ht29 colonic carcinoma cells.

Cl- secretion in HT29 cells is regulated by agonists such as carbachol, neurotensin and adenosine 5'-triphosphate (ATP). These agonists induce Ca2+ store release as well as Ca2+ influx from the extracellular space. The increase in cytosolic Ca2+ enhances the Cl- and K+ conductances of these cells. Removal of extracellular Ca2+ strongly attenuates the secretory response to the above-mentioned agonists. The present study utilises patch-clamp methods to characterise the Ca2+ influx pathway. Inhibitors which have been shown previously to inhibit non-selective cation channels, such as flufenamate (0.1 mmol.l-1, n = 6) and Gd3+ (10 micromol.l-1, n=6) inhibited ATP (0.1 mmol.l-1) induced increases in whole-cell conductance (Gm). When Cl- and K+ currents were inhibited by the presence of Cs2SO4 in the patch pipette and gluconate in the bath, ATP (0.1 mmol.l-1) still induced a significant increase in Gm from 1.2 +/- 0.3 nS to 4.7 +/- 1 nS (n = 24). This suggests that ATP induces a cation influx with a conductance of approximately 3-4 nS. This cation influx was inhibited by flufenamate (0.1 mmol.l-1, n = 6) and Gd3+ (10 micromol.l-1, n = 9). When Ba2+ (5 mmol.l-1) and 4,4'-diisothiocyanato-stilbene-2-2'-disulphonic acid (DIDS, 0.1 mmol.l-1) were added to the KCl/K-gluconate pipette solution to inhibit K+ and Cl- currents and the cells were clamped to depolarised voltages, ATP (0.1 mmol.l-1) reduced the membrane current (Im) significantly from 86 +/- 14 pA to 54 +/- 11 pA (n = 13), unmasking a cation inward current. In another series, the cation inward current was activated by dialysing the cell with a KCl/K-gluconate solution containing 5-10 mmol.l-1 1,2-bis-(2-aminoethoxy)ethane-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis-(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA). The zero-current membrane voltage (Vm) and Im (at a clamp voltage of +10 mV) were monitored as a function of time. A new steady-state was reached 30-120 s after membrane rupture. Vm depolarised significantly from -33 +/- 2 mV to -12 +/- 1 mV, and Im fell significantly from 17 +/- 2 pA to 8.9 +/- 1.0 pA (n = 71). This negative current, representing a cation inward current, was activated when Ca2+ stores were emptied and was reduced significantly ( Im) when Ca2+ and/or Na+ were removed from the bathing solution: removal of Ca2+ in the absence of Na+ caused a Im of 5.0 +/- 1.2 pA (n = 12); removal of Na+ in the absence of Ca2+ caused a Im of 12.8 +/- 3.5 pA (n = 4). The cation inward current was also reduced significantly by La3+, Gd3+, and flufenamate. We conclude that store depletion induces a Ca2+/Na+ influx current in these cells. With 145 mmol.l-1 Na+ and 1 mmol.l-1 Ca2+, both ions contribute to this cation inward current. This current is an important component in the agonist-regulated secretory response.

Cytosolic Ca2+ dynamics in hamster ascending thin limb of Henle's loop

Intracellular calcium plays an important role in the regulation of Cl- reabsorption in the ascending thin limb of Henle's loop (ATL). To elucidate the cytosolic Ca2+ dynamics in the ATL, intracellular Ca2+ concentration activity ([Ca2+]i) was measured in the in vitro microperfused hamster ATL using fura 2. Basal [Ca2+]i was 89.1 +/- 7.3 nM (n = 9 tubules). Removal of Ca2+ from the peritubular solution decreased [Ca2+]i from 89.1 +/- 7.3 to 64.1 +/- 7.1 nM in 2 min (n = 9, P < 0.05), whereas [Ca2+]i did not change after removal of Ca2+ from the luminal solution. Addition of 1 mM NaCN to the bath increased [Ca2+]i. This effect was completely abolished by the elimination of ambient Ca2+. Trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) in the bath reversibly increased [Ca2+]i, whereas addition of 1 mM ouabain to the bath decreased [Ca2+]i. Rates of changes in [Ca2+]i after removal and replacement of basolateral Ca2+ were not affected by removal of Na+, K+, or Cl- from the bath, whereas nicardipine decreased these parameters. Increasing bath K+ from 5 to 100 mM decreased [Ca2+]i from 69.3 +/- 5.8 to 50.8 +/- 5.0 nM in 1 min (n = 6, P < 0.05). Subsequent reduction of K+ from 100 to 5 mM increased [Ca2+]i to 174.0 +/- 30.8 nM in 1 min, followed by a gradual decrease in [Ca2+]i to a steady-state level of 74.2 +/- 8.0 nM in 2 min. Changes in basolateral K+ concentration did not affect [Ca2+]i in the absence of ambient Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Na/Ca exchange in the basolateral membrane of the A6 cell monolayer: role in Cai homeostasis.

The presence of a Na/Ca exchanger in A6 cells was investigated by measuring intracellular calcium (Cai) fluctuations and the 45Ca fluxes through the basolateral membranes (blm) of the cell monolayer. Removal of Na+ from the medium produced a transient increase in Cai followed by a regulatory phase returning Cai to control levels in 3-4 min, this phase being greatly accelerated (< 60 s) by NaCl addition (apparent Km of approximately 5 mM Na+). The Cai increase was only found with the Na(+)-free medium on the basolateral side of the cell monolayer. A twofold increase in the 45Ca influx was observed under these conditions. In Ca(2+)- depleted cells, the initial Cai increase after Ca2+ addition to the medium was greater when the putative Na/Ca exchanger was not functioning (i.e. in a Na(+)-free medium). 45Ca effluxes through the blm of the monolayer were greatly and transiently increased by a Na(+)-free medium on the serosal side and blocked by orthovanadate (1 mM). The Cai increased induced by a hypo-osmotic shock was greater in cells bathed in a Na(+)-medium, conditions expected to block the activity of the Na/Ca exchanger. These findings support the hypothesis that a Na/Ca exchanger is present on the blm of A6 cells and affirm its role in Cai homeostasis in steady-state conditions and following osmotic shock. In addition, a Ca2+ pump also located on the blm and Ca2+ stores sensitive to inositol 1,4,5-trisphosphate were found to be implicated in Cai homeostasis.

The influence of calcium transients on intracellular pH in cortical neurons in primary culture

The objective of this study was to assess the influence of Ca 2+ influx on intracellular pH (pH i) of neocortical neurons in primary culture. Neurons were exposed to glutamate (100–500 μM) or KCl (50 mM), and pH i was recorded with microspectroflurometric techniques. Additional experiments were carried out in which calcium influx was triggered by ionomycin (2 μM) or the calcium ionophore 4-Br-A23187 (2 μM). Glutamate exposure either caused no, or only a small decrease in pH i (ΔpH ≈ 0.06 units). When a decrease was observed, a rebound rise in pH i above control was observed upon termination of glutamate exposure. In about 20% of the cells, the acidification was more pronounced (ΔpH ≈ 0.20 units), but all these cells had high control pH i values, and showed gradual acidification. Exposure of cells to 50 mM KCl consistently increased pH i. Since this increase was similar in the presence and nominal absence of HCO 3 −, it probably did not reflect influx of HCO 3 − via a Na +-HCO 3 − symporter. Furthermore, since it occurred in the absence of external Ca 2+ (or a measurable rise in Ca i 2+) it seemed independent of Ca 2+ influx. It is tentatively concluded that the rise in pH i was due to reduced passive influx of H + along the electrochemical gradient, which is reduced by depolarization. In Ca 2+-containing solutions, depolarization led to a rebound increase in pH i above control. This, and the rebound found after glutamate transients, may reflect Ca 2+-triggered phosphorylation and upregulation of the Na +/H + antiporter which extrudes H + from the cell. Ionomycin and 4-Br-A23187 gave rise to a large rise in Ca i 2+ and to alkalinization of the cell (ΔpH ≈ 0.5). Since amiloride or removal of Na + from the external solution did not alter the rise in pH i, it was probably not due to accelerated H + extrusion. However, removal of Ca 2+ from extracellular fluid prevented the rise, suggesting that it was secondary to Ca 2+/2H + exchange across plasma membranes.

Diacylglycerol-induced activation of protein kinase C attenuates Na+ currents by enhancing inactivation from the closed state.

The causes of attenuation of Na+ currents by diacylglycerol (DAG)-induced protein kinase C (PKC) activation in mouse neuroblastoma N1E-115 cells were investigated using the cell-attached patch, and the perforated-patch (nystatin based) whole-cell voltage-clamp techniques. Activation of PKC by DAG attenuated Na+ currents. Attenuation occurred in the absence of significant changes in the time-course of Na+ currents. However, the steady-state inactivation curve of these currents shifted to more negative voltages by approximately 20 mV. Here we demonstrate that the time-course of inactivation is accelerated by treatment with DAG-like substances in a voltage-dependent manner (time constant of inactivation decreased by 2- and 3.6-fold at -60, and -30 mV, respectively). In cell-attached patches, treatment with DAG compounds increased the percentage of current traces showing no single Na+ channel openings in response to depolarizing voltage-clamp pulses. Moreover, the average of current traces containing single Na+ channel openings was essentially the same in control conditions and after treatment with DAG compounds. Removal of Na+ channel inactivation by the alkaloid batrachotoxin prevented the attenuation of Na+ currents by PKC activation via DAGs. Taken together, these data strongly suggest that PKC-induced attenuation of Na+ currents is linked to an enhancement of Na+ channel inactivation. This attenuation is caused by an increase in the number of Na+ channels inactivating directly from the closed state(s). This inactivation pathway represents a simple and efficient physiological mechanism by which PKC activation might modulate the electrical activity of excitable cells.

Ammonia stimulates the release of taurine from cultured astrocytes

The effect of ammonia on the release of the neuroactive amino acids taurine (TAU), γ-aminobutyric acid (GABA) and d-aspartate ( d-ASP), an analog of l-glutamate ( l-GLU), from cultured rat cortical astrocytes was studied. NH 4Cl (1 and 5 mM) induced the release of TAU. TAU release was reduced when Na + was removed, and was almost completely abolished when Cl − was omitted. In contrast, TAU basal release was enhanced upon removal of Na + or Cl −. Ammonia inhibited the release of GABA and d-ASP. Ammonia-induced release of astroglial TAU may modify the neuronal excitability accompanying hyperammonemic conditions.

Relationship between intra- and extracellular pH in primary cultures of rat astrocytes

We studied the influence of extracellular pH (pHe) on the mechanisms regulating intracellular pH (pHi) in astrocytes cultured from neonatal rat cortex, using single cell microspectrofluorometry and the pH-sensitive fluorophore 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. When pHe was maintained at control values of 7.35 during acid transients caused by an increased CO2 tension, pHi was rapidly regulated back to normal. However, at pHe 6.9 or below, there was no recovery of pHi. Steady-state pHi was also strongly dependent on pHe (pHi = 1.14 + 0.80 pHe). The pHi recovery after normalization of pHe was very rapid, indicating that a prolonged exposure to a low pH stimulates pH-regulating mechanisms, and was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and amiloride or the removal of Na+. Recovery was also slowed down by Cd2+. The pHe-dependent acidification was not critically influenced by DIDS or amiloride and was not inhibited by tetrodotoxin, tetraethylammonium, Ba2+,2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline, kynurenic acid, 5-hydroxysaclofen, bicuculline, Cd2+, or albumin. The results emphasize the importance of pHe for pHi regulation and suggest that in astrocytes pHi is not regulated back to normal levels until pHe is normalized.

Membrane localization of H+ and HCO3- transporters in the rat pancreatic duct.

The pancreatic duct secretes alkaline fluid that is rich in HCO3- and poor in Cl-. The molecular mechanisms that mediate ductal secretion and are responsible for the axial gradients of Cl- and HCO3- along the ductal tree are not well understood because H+ and HCO3- transport by duct cells have not been characterized or localized. To address these questions, we microdissected the intralobular, main, and common segments of the rat pancreatic duct. H+ and HCO3- transporters were characterized and localized by following intracellular pH while perfusing the bath and the lumen of the ducts. In intralobular ducts, Na(+)-dependent and amiloride-sensitive recovery from acid load in the absence of HCO3- was used to localize a Na+/H+ exchanger to the basolateral membrane (BLM). Modification of Cl- gradients across the luminal (LM) and BLM in the presence of HCO3- showed the presence of Cl-/HCO3- exchangers on both membranes of intralobular duct cells. Measurement of the effect of Cl- on one side of the membrane on the rate and extent of pHi changes caused by removal and addition of Cl- to the opposite side suggested that both exchangers are present in the same cell. In the presence of HCO3-, intralobular duct cells used three separate mechanisms to extrude H+: (a) BLM-located Na+/H+ exchange, (b) Na(+)-independent vacuolar-type H+ pump, and (c) BLM-located, Na(+)-dependent, amiloride-insensitive, and 4',4'-diisothiocyanatostilbene-2,2'-disulfonic acid sensitive mechanism, possibly a Na(+)-dependent HCO3- transporter. The main and common segments of the duct displayed similar mechanisms and localization of H+ and HCO3- transporters to the extent studied in the present work. In addition to the transporters found in intralobular ducts, the main and common ducts showed Na+/H+ exchange activity in the LM. Three tests were used to exclude a significant luminal to basolateral Na+ leak as the cause for an apparent luminal Na+/H+ exchange in an HCO3- secreting cells: (a) addition of amiloride and removal of Na+ from the LM had a profound effect on Na+/H+ exchange activity on the BLM and vice versa; (b) inhibition of all transporters in the BLM by bathing the duct in the inert hydrocarbon Fluorinert FC-75 did not prevent cytosolic acidification caused by removal of luminal Na+; and (c) luminal Na+ did not activate the basolateral Na(+)-dependent HCO3- transporter. An Na(+)-independent, bafilomycin-sensitive H+ pumping activity was marginal in the absence of HCO3-.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular pH regulation by the plasma membrane V-ATPase in Malpighian tubules of Drosophila larvae.

The functional significance of the apical vacuolar-type proton pump (V-ATPase) in Drosophila Malpighian tubules was studied by measuring the intracellular pH (pHi) and luminal pH (pHlu) with double-barrelled pH-microelectrodes in proximal segments of the larval anterior tubule immersed in nominally bicarbonate-free solutions (pHo 6.9). In proximal segments both pHi (7.43 +/- 0.20) and pHlu (7.10 +/- 0.24)) were significantly lower than in distal segments (pHi 7.70 +/- 0.29, pHlu 8.09 +/- 0.15). Steady-state pHi of proximal segments was much less sensitive to changes in pHo than pH of the luminal fluid (delta pHlu/delta pHo was 0.49 while delta pHi/delta pHo was 0.18; pHo 6.50-7.20). Re-alkaliniziation from an NH4Cl-induced intracellular acid load (initial pHi recovery rate 0.55 +/- 0.34 pH.min-1) was nearly totally inhibited by 1 mmol.l-1 KCN (96% inhibition) and to a large degree (79%) by 1 mumol.l-1 bafilomycin A1. In contrast, both vanadate (1 mmol.l-1) and amiloride (1 mmol.l-1) inhibited pHi recovery by 38% and 33%, respectively. Unlike amiloride, removal of Na+ from the bathing saline had no effect on pHi recovery, indicating that a Na+/H+ exchange is not significantly involved in pHi regulation. Instead pHi regulation apparently depended largely on the availability of ATP and on the activity of the bafilomycin-sensitive proton pump.

Evidence for a Sodium-Dependent Calcium Influx in Isolated Rat Hepatocytes Undergoing ATP Depletion

ATP depletion caused by menadione and triethyllead in isolated hepatocytes is associated with intracellular acidosis and a sustained increase in intracellular Na + and Ca 2+ concentrations. Removal of Na + from the incubation medium as well as the inclusion of EGTA largely prevented the increase in cytosolic Ca 2+, thus indicating that Ca 2+ was mobilized from the extracellular medium in response to Na + load. To further validate these findings, hepatocytes were incubated with a combination of sodium propionate and ouabain in order to induce intracellular acidosis and inhibit Na + extrusion. This treatment promoted a marked increase in intracellular Na + and Ca 2+ concentrations that was prevented by omission of Na + from the incubation medium as well as by agents that inhibited cellular Na + influx. These data indicate that following Na + load, Ca 2+ can be accumulated in hepatocytes via a Na +/Ca 2+ antiporter operating on a reverse mode.