Mixed type inhibition of the renal Na+/H+ antiporter by Li+ and amiloride. Evidence for a modifier site

We studied the interactions of Na+, Li+, and amiloride on the Na+/H+ antiporter in brush-border membrane vesicles from rabbit renal cortex. Cation-mediated collapse of an outwardly directed proton gradient (pHin = 6.0; pHout = 7.5) was monitored with the fluorescent amine, acridine orange. Proton efflux resulting from external addition of Na+ or Li+ exhibited simple saturation kinetics with Hill coefficients of 1.0. However, kinetic parameters for Na+ and Li+ differed (Km for Li+ = 1.2 +/- 0.1 mM; Km for Na+ = 14.3 +/- 0.8 mM; Vmax for Li+ = 2.40 +/- 0.07 fluorescence units/s/mg of protein; Vmax for Na+ = 7.10 +/- 0.24 fluorescence units/s/mg of protein). Inhibition of Na+/H+ exchange by Li+ and amiloride was also studied. Li+ inhibited the Na+/H+ antiporter by two mechanisms. Na+ and Li+ competed with each other at the cation transport site. However, when [Na+] was markedly higher than [Li+], [( Na+] = 90 mM; [Li+] less than 1 mM), we observed noncompetitive inhibition (Vmax for Na+/H+ exchange reduced by 25%). The apparent Ki for this noncompetitive inhibition was congruent to 50 microM. In addition, 2-30 mM intravesicular Li+, but not Na+, resulted in trans inhibition of Na+/H+ exchange. Amiloride was a mixed inhibitor of Na+/H+ exchange (Ki = 30 microM, Ki' = 90 microM) but was only a simple competitive inhibitor of Li+/H+ exchange (Ki = 10 microM). At [Li] = 1 mM and [amiloride] less than 100 microM, inhibition of Na+/H+ exchange by a combination of the two inhibitors was always less than additive. These results suggest the presence of a cation-binding site (separate from the cation-transport site) which could be a modifier site of the Na+/H+ antiporter.     

Arachidonic acid metabolites by cytochrome P-450 dependent monooxygenase pathway in bovine adrenal fasciculata cells

[1-14C]Arachidonic acid was incubated with microsomes of bovine adrenal fasciculata cells in the presence of 1 mM NADPH for 30 min at 37 degrees C. The metabolites were separated and purified by reverse phase high performance liquid chromatography, and identified by gas chromatography-mass spectrometry. Identified metabolites were four dihydroxyeicosatrienoic acids (DHTs) (5,6-, 8,9-, 11,12-, 14,15-DHTs), 20-hydroxyeicosatetraenoic acid and eicosatetradioic acid. The formation of these metabolites was dependent on NADPH and inhibited by SKF-525A. 14,15-DHT was also formed by isolated bovine adrenal fasciculata cells. These results indicate that cytochrome P-450 dependent arachidonate monooxygenase pathway may exist in bovine adrenal fasciculata cells. Addition of the chemically synthesized epoxyeicosatrienoic acids (EETs) to isolated bovine adrenal fasciculata cells stimulated cortisol production. Among four regioisomeric EETs, 14,15-EET was most potent and stimulated steroidogenesis in a dose-related manner over a range of 0.5 to 5.0 microM.     

Sodium-dependent regulation of steroidogenesis in rat granulosa cells: possible involvement of a Na+/H+ antiport

The possible role of Na+/H+ antiport in the gonadotropic regulation of steroidogenesis was examined in rat granulosa cells incubated for up to 6 h in a chemically defined medium in the absence or presence of Na+ (128 mM), gonadotropin (FSH or LH; 0-500 ng/ml), dibutyryl cyclic AMP [Bu)2cAMP; 2 mM) and amiloride (0-1 mM). Replacement of Na+ (Na+0) in the incubation medium with choline chloride resulted in a marked decrease in basal and LH-, FSH- and (Bu)2cAMP-stimulated progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) synthesis in vitro. The Na+/H+ exchange inhibitor, amiloride significantly suppressed basal and hormone-stimulated progestin production dose-dependently in the presence of Na+0. However, it was without effect in Na+-deficient medium. The effect of the inhibitor on progestin production appeared to be directed at specific step(s) involved in the synthesis of pregnenolone, as concentrations of amiloride which inhibited progesterone production failed to influence the metabolism of exogenous pregnenolone to progestins. Cell viability and the incorporation of [3H]leucine into acid-precipitable material were not affected by amiloride. Our findings support the contention that extracellular sodium is important for steroidogenesis in rat granulosa cells. The inhibition by amilordie indicates an involvement of the Na+/H+ exchange in the regulation of this granulosa cell function.     

Polarized distribution of the Na+/H+ exchange system in a renal cell line (LLC-PK1) with characteristics of proximal tubular cells

Studies of Na+ and H+ transport by confluent monolayers of the epithelial cell line LLC-PK1 were performed to verify the presence of a Na+/H+ exchange system. The presence of an outwardly directed H+ gradient produced a large stimulation of Na+ influx measured under net flux conditions. Amiloride (10(-3) M) completely inhibited Na+ influx stimulated by the H+ gradient and part of the Na+ influx measured in the absence of a pH gradient. Half-maximal inhibition of the Na+ influx stimulated by a pH gradient at 143 mM Na was observed at 5 microM amiloride. The presence of an inwardly oriented proton gradient also stimulated Na+ efflux from Na+-loaded cells. The stimulation was completely inhibited by the presence of 10(-3) M amiloride in the washout medium. These results indicate that this system could operate in the opposite direction depending on the orientation of the Na+ and H+ gradient. Incubation in Na+-free medium or in the presence of 10(-3) M ouabain resulted in a dramatic decrease of H+ release from LLC-PK1 cells. This H+ release was largely, although not completely, inhibited by 10(-4) M amiloride. Neither chloride substitution by the impermeable anion isethionate nor incubation in the presence of the ionophore valinomycin in high K+ medium affected Na+ influx by stimulated by a pH gradient. Inhibition of the Na+ influx by amiloride occurred only from the apical side of the monolayer. These results indicate that the Na+/H+ exchange system in LLC-PK1 monolayers is specifically localized in the apical membrane of the epithelial cells.     

Activation of JNK/c-Jun is required for the proliferation, survival, and angiogenesis induced by EET in pulmonary artery endothelial cells

Pulmonary artery endothelial plexiform lesion is responsible for pulmonary vascular remodeling (PVR), a basic pathological change of pulmonary arterial hypertension (PAH). Recent evidence suggests that epoxyeicosatrienoic acid (EET), which is derived from arachidonic acid by cytochrome p450 (CYP) epoxygenase, has an essential role in PAH. However, until now, most research has focused on pulmonary vasoconstriction; it is unclear whether EET produces mitogenic and angiogenic effects in pulmonary artery endothelial cells (PAEC). Here we found that 500 nM/l 8,9-EET, 11,12-EET, and 14,15-EET markedly augmented JNK and c-Jun activation in PAECs and that the activation of c-Jun was mediated by JNK, but not the ERK or p38 MPAK pathway. Moreover, treatment with 8,9-EET, 11,12-EET, and 14,15-EET promoted cell proliferation and cell-cycle transition from the G0/G1 phase to S phase and stimulated tube formation in vitro. All these effects were reversed after blocking JNK with Sp600125 (a JNK inhibitor) or JNK1/2 siRNA. In addition, the apoptotic process was alleviated by three EET region isomers through the JNK/c-Jun pathway. These observations suggest that 8,9-EET, 11,12-EET, and 14,15-EET stimulate PAEC proliferation and angiogenesis, as well as protect the cells from apoptosis, via the JNK/c-Jun pathway, an important underlying mechanism that may promote PAEC growth and angiogenesis during PAH.     

The mechanism of insulin stimulation of (Na+,K+)-ATPase transport activity in muscle

Since the mechanism underlying the insulin stimulation of (Na+,K+)-ATPase transport activity observed in multiple tissues has remained undetermined, we have examined (Na+,K+)-ATPase transport activity (ouabain-sensitive 86Rb+ uptake) and Na+/H+ exchange transport (amiloride-sensitive 22Na+ influx) in differentiated BC3H-1 cultured myocytes as a model of insulin action in muscle. The active uptake of 86Rb+ was sensitive to physiological insulin concentrations (1 nM), yielding a maximum increase of 60% without any change in 86Rb+ permeability. In order to determine the mechanism of insulin stimulation of (Na+,K+)-ATPase activity, we demonstrated that insulin also stimulates passive 22Na+ influx by Na+/H+ exchange transport (maximal 200% increase) and an 80% increase in intracellular Na+ concentration with an identical time course and dose-response curve as insulin-stimulated (Na+,K+)-ATPase transport activity. Incubation of the cells with high [Na+] (195 mM) significantly potentiated insulin stimulation of ouabain-inhibitable 86Rb+ uptake. The ionophore monensin, which also promotes passive Na+ entry into BC3H-1 cells, mimics the insulin stimulation of ouabain-inhibitable 86Rb+ uptake. In contrast, incubation with amiloride or low [Na+] (10 mM), both of which inhibit Na+/H+ exchange transport, abolished the insulin stimulation of (Na+,K+)-ATPase transport activity. Furthermore, each of these insulin-stimulated transport activities displayed a similar sensitivity to amiloride. These results indicate that insulin stimulates a large increase in Na+/H+ exchange transport and that the resulting Na+ influx increases the intracellular Na+ concentration, thus activating the internal Na+ transport sites of the (Na+,K+)-ATPase. This Na+ influx is, therefore, the mediator of the insulin-induced stimulation of membrane (Na+,K+)-ATPase transport activity classically observed in muscle.     

Effect of Protein Kinase C Modulators on 14,15-Epoxyeicosatrienoic Acid Incorporation into Astroglial Phospholipids

Abstract: Our previous studies have shown that 14,15-epoxyeicosatrienoic acid (14,15-EET) is a major product of arachidonic acid metabolism in astrocytes. The purpose of this study was to investigate cellular regulation of 14,15-EET incorporation, distribution, and metabolism in primary cultures of rat brain cortical astrocytes. Incorporation of 14,15-EET into astrocytes was lower (93,390 ± 11,121 dpm/5 × 10⁶ cells) than incorporation of 8,9-EET (226,500 ± 5,567 dpm/5 × 10⁶ cells) and arachidonic acid (321,600 ± 1,200 dpm/5 × 10⁶ cells). 14,15-EET was distributed in the order neutral lipids and free fatty acids (solvent front) ? phosphatidylcholine (PC) > phosphatidylinositol (PI) > phosphatidylethanolamine. In contrast, 8,9-EET and arachidonic acid were exclusively incorporated into PC. During incubation, astroglial epoxide hydrolase selectively metabolized 14,15-EET, but not 8,9-EET, to its vic-diol. Although 4-phenylchalcone oxide, a potent inhibitor of epoxide hydrolase, completely inhibited 14,15-EET metabolism, a large amount of cell-incorporated radioactivity remained as free 14,15-EET. Long-term exposure of astrocytes to 4β-phorbol 12-myristate 13-acetate (4β-PMA) resulted in a time-dependent incorporation of 14,15-EET into PI but not in control cells exposed to 4α-phorbol 12,13-didecanoate. PKC down-regulation completely inhibited epoxide hydrolase metabolism of 14,15-EET. Following recovery of down-regulated PKC, 1 week after treatment with 4β-PMA, astrocytes regained their normal pattern of low incorporation of 14,15-EET. Protein kinase C (PKC) inhibition by staurosporine enhanced 14,15-EET incorporation without affecting its metabolism to 14,15-dihydroxyeicosatrienoic acid. Incorporation of 14,15-EET by PKC-down-regulated cells was inhibited by thimerosal, a known inhibitor of fatty acyl-CoA synthase. Our results suggest that the lower incorporation of 14,15-EET into astroglial cells may be due to modulation of PKC-mediated cellular mechanism(s).     

Activation of the Na+/H+ and Cl-/HCO3- exchange by stimulation of acid secretion in the parietal cell

Upon stimulation, the gastric parietal cell secretes a large quantity of isotonic HCl across its apical membrane which must be accompanied by the generation of base in the cytosol. The ability of this cell type to regulate cytosolic pH (pHi) was examined as a function of stimulation of acid secretion by histamine or forskolin. The pHi was estimated from the change of fluorescence of the trapped dye, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-bis-carboxyethylcarbo xy fluorescein in a purified cell suspension of rabbit parietal cells. Stimulation of the cell suspension raised pHi by an average of 0.13 +/- 0.038 pH units. The H+,K+-ATPase inhibitor, SCH28080 (2-methyl-8-[phenyl-methoxy]-imidazo-(1,2)-pyridine-3-acetonitrile) had only a small effect on the increase of pHi, therefore, was largely independent of H+,K+-ATPase activity. In Na+-free medium, where Na+/H+ exchange would be absent, the rise of pHi was only 0.03 pH units. This increase was blocked by SCH28080, showing that this small increment was the result of acid secretion. In Na+-containing medium, 90% of the increase was inhibited by an inhibitor of Na+/H+ exchange, dimethyl amiloride (DMA). This compound also blocked changes in pHi due to changes in extracellular Na+. Accordingly, most of the change in pHi upon stimulation of acid secretion by histamine and forskolin is due to activation of Na+/H+ exchange in the parietal cell basal-lateral membrane. The addition of DMA to stimulated, but not resting cells, gave a rapid acidification that was blocked by inhibition of anion exchange by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), showing that anion exchange was also activated by stimulation. In single cell recording, canalicular and cytosolic pH were monitored simultaneously using 9-amino acridine and dimethyl carboxyfluorescein, respectively. Cytosolic alkalinization correlated with acid accumulation in the secretory canaliculus until a set point was reached. Thereafter, acidification continued without further change in pHi. To determine the role of Na+/H+ and Cl-/HCO3- exchange in acid secretion, Cl(-)-depleted cells were suspended in medium containing 40 mM Cl-. DMA and DIDS each blocked acid secretion by about 40%, but in combination, acid secretion was blocked by more than 90%. Thus, basal-lateral Na+/H+ and Cl-/HCO3- exchange activities are necessary for acid secretion across the apical membrane of the parietal cell.     

Kinetic characteristics of 22Na transport in human and rat erythrocytes during cytoplasm acidification and cell compression

Under normal conditions (pH0 = 7.4, pHi = 7.1-7.2) amiloride, a Na+/H+ exchange inhibitor, does not influence Na+ intake by human and rat erythrocytes. Acidification of the cytoplasm (pHi approximately 6.4) is accompanied by the acceleration of 22Na intake, which is decreased after addition of 1 mM amiloride (by 50 and 80%, respectively). The Ki value of amiloride for human and rat erythrocytes is 30 and 250 microM, respectively. In rat erythrocytes the dependence of the rate of the delta pH-induced incorporation of 22Na on Na+ concentration is described by a saturation curve (K0.5 for Na0+ is approximately 40 mM), whereas in human erythrocytes it obeys the diffusion kinetics. These results suggest that the Na+/H+ exchange takes place in rat erythrocytes, but is absent in human erythrocytes. In rat erythrocytes the Na+/H+ exchange can be induced by cell compression which can be caused either by decreasing the KCl content (after addition of valinomycin) or by increasing the osmolarity of the medium (in the presence of sucrose). The rate of Na+/H+ exchange induced by cell compression is increased by 60-70% after addition of protein kinases A and C activators. No effect of intracellular Ca2+ on the rate of the Na+/H+ exchange in rat erythrocytes is observed.     

Pathways of epoxyeicosatrienoic acid metabolism in endothelial cells. Implications for the vascular effects of soluble epoxide hydrolase inhibition

Epoxyeicosatrienoic acids (EETs) are products of cytochrome P-450 epoxygenase that possess important vasodilating and anti-inflammatory properties. EETs are converted to the corresponding dihydroxyeicosatrienoic acid (DHET) by soluble epoxide hydrolase (sEH) in mammalian tissues, and inhibition of sEH has been proposed as a novel approach for the treatment of hypertension. We observed that sEH is present in porcine coronary endothelial cells (PCEC), and we found that low concentrations of N,N'-dicyclohexylurea (DCU), a selective sEH inhibitor, have profound effects on EET metabolism in PCEC cultures. Treatment with 3 microM DCU reduced cellular conversion of 14,15-EET to 14,15-DHET by 3-fold after 4 h of incubation, with a concomitant increase in the formation of the novel beta-oxidation products 10,11-epoxy-16:2 and 8,9-epoxy-14:1. DCU also markedly enhanced the incorporation of 14,15-EET and its metabolites into PCEC lipids. The most abundant product in DCU-treated cells was 16,17-epoxy-22:3, the elongation product of 14,15-EET. Another novel metabolite, 14,15-epoxy-20:2, was present in DCU-treated cells. DCU also caused a 4-fold increase in release of 14,15-EET when the cells were stimulated with a calcium ionophore. Furthermore, DCU decreased the conversion of [3H]11,12-EET to 11,12-DHET, increased 11,12-EET retention in PCEC lipids, and produced an accumulation of the partial beta-oxidation product 7,8-epoxy-16:2 in the medium. These findings suggest that in addition to being metabolized by sEH, EETs are substrates for beta-oxidation and chain elongation in endothelial cells and that there is considerable interaction among the three pathways. The modulation of EET metabolism by DCU provides novel insight into the mechanisms by which pharmacological or molecular inhibition of sEH effectively treats hypertension.     

Interaction of guanidinium and guanidinium derivatives with the Na+/H+ exchange system

Guanidinium, a small organic monovalent cation that is permeant through voltage-dependent cationic channels cannot be transported by the cardiac Na+/H+ exchange system. Yet it recognizes the exchanger and is able to block its activity (K0.5 = 30 mM). Guanidinium derivatives that do not belong to the amiloride series and which possess potent antihypertensive properties also block the activity of the Na+/H+ exchange system in various cell types with a greater potency than unsubstituted guanidinium. The most potent compound found, guanochlor, has an affinity for the exchanger ranging between 0.5 microM and 6 microM in different systems and is more potent than amiloride in all systems studied. Guanochlor has the same action as amiloride derivatives on the cardiac cells; it prevents intracellular pH recovery in cardiac cells that have been acidified and also antagonizes the effect of ouabain on 45Ca2+ uptake by chick cardiac cells. Guanochlor does not compete with [3H]ethylpropylamiloride for its binding to the Na+/H+ exchange system of rabbit kidney brush border membrane. It is suggested that guanochlor recognizes a binding site on the Na+/H+ exchanger that is distinct from the amiloride binding site.     

Collisionally induced dissociation of epoxyeicosatrienoic acids and epoxyeicosatrienoic acid-phospholipid molecular species.

Four isomers of epoxyeicosatrienoic acid (EET) can be formed by cytochrome P-450 oxidation of arachidonic acid: 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid. The collision-induced dissociation of the [M-H]- anion at m/z 319 from each of these isomers, using negative-ion fast atom bombardment ionization and a triple quadrupole mass spectrometer, resulted in a series of common ions as well as ions characteristic of each isomer. The common ions were m/z 301 [M-H2O]- and 257 [M-(H2O + CO2)]-. Unique ions resulted from cleavages alpha to the epoxide moiety to form either conjugated carbanions or aldehydes. Mechanisms involving charge site transfer are suggested for the origin of these ions. A distonic ion series that may involve a charge-remote fragmentation mechanism was also observed. The epoxyeicosatrienoic acids were also incorporated into cellular phospholipids following incubation of the free acid with murine mast cells in culture. Negative fast atom bombardment mass spectrometry of purified glycerophosphoethanolamine-EET species and glycerophosphocholine-EET species yielded abundant [M-H]- and [M-CH3]- ions, respectively. The collision-induced dissociation of these specific high-mass ions revealed fragment ions characteristic of the epoxyeicosatrienoic acids incorporated (m/z 319, 301, and 257) and the same unique ions as those seen with each isomeric epoxyeicosatrienoic acid. With this direct method of analysis, phospholipids containing the four positional isomers of EET, including the highly labile (5,6-EET), could be identified as unique molecular species in mast cells incubated with EET.     

Sodium/Proton Exchange in Cultured Bovine Adrenal Medullary Cells

We investigated the presence of Na+/H+ exchange in cultured bovine adrenal medullary cells. The intracellular pH in control cells measured by 5,5-dimethyl[2-14C]oxazolidine-2,4-dione was 7.13 +/- 0.02 (n = 6). Removal of Na+ from the incubation medium shifted the intracellular pH down to 6.67 +/- 0.12 (n = 6). Reintroduction of Na+ to the medium caused a rapid recovery in intracellular pH to 7.20-7.30 that was associated with an increase in uptake of 22Na+ by the cells. Both increases in intracellular pH and uptake of 22Na+ were inhibited by amiloride, an inhibitor of Na+/H+ exchange. The recovery of intracellular pH by addition of Na+ was partially inhibited by quinidine, another inhibitor of Na+/H+ exchange, but not by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, an anion-exchange (Cl-/HCO3-) inhibitor. Li+ could substitute for Na+ in the recovery of intracellular pH. Carbachol caused an increase in intracellular pH from 7.12 +/- 0.01 to 7.21 +/- 0.02 (n = 10). This increase in intracellular pH caused by carbachol was inhibited by amiloride. These results suggest the existence of an amiloride-sensitive Na+/H+ exchange that regulates the intracellular pH in adrenal medullary cells.     

Na+/H+ exchange activation mediates the lipopolysaccharide-induced proliferation of human B lymphocytes and is impaired in malignant B-chronic lymphocytic leukemia lymphocytes

In various mammalian cell types the stimulation of the plasma membrane amiloride-sensitive Na+/H+ exchange and the resulting increase of intracellular pH (pHi) play a key role in the initiation of cell proliferation. In the present work we have investigated whether Na+/H+ exchange is involved in normal human B cell proliferation and whether it is also operating in malignant B-chronic lymphocytic leukemia (B-CLL) lymphocytes. Our results show that: 1) normal human B cells contain an operating Na+/H+ exchanger, as inferred by their ability to recover pHi after acid-loading in a HCO3- -free medium and by evidences that LPS and phorbol ester PMA elicit a pHi rise inhibitable by either 5-(N-ethyl-N-isopropyl)amiloride (EIPA) or a Na+-free medium; 2) LPS-induced proliferation of normal human B cells is strongly inhibited when the amiloride analog EIPA (5 microM) is present in the culture medium (after 72 h the proportion of B cells incorporation bromodeoxyuridine falls from 13.9 +/- 3.9% to 2.8 +/- 1.1%); 3) EIPA does not affect BdR incorporation when B cells proliferation is induced by the co-mitogenic activity of IL-4 and low m.w. B cell growth factor (BCGF); 4) B-CLL cells, which proliferate in response to IL-4/BCGF but not to LPS, fail to increase pHi above their pHi resting levels when challenged with LPS or PMA and pHi recovery after acid-loading is highly impaired. These results lead to conclude that Na+/H+ exchange operation is necessary for LPS-(but not for IL-4/BCGF)-induced proliferation of human normal B lymphocytes and that Na+/H+ exchange activation is impaired in malignant B-CLL lymphocytes.     

Serum regulates Na+/H+ exchange in Caco-2 cells by a mechanism which is dependent on F-actin.

Regulation of Na+/H+ exchange by fetal bovine serum was studied in Caco-2 cells, an established cell line derived from a human colon carcinoma. Cells were grown as polarized monolayers on collagen-coated filters and intracellular pH measured fluorometrically with 2',7'-bis(2-carboxymethyl)-5,6-carboxyfluorescein. Na+/H+ exchange was reduced 64% when cells were deprived of serum for 4 h. In contrast to other cell types, readdition of serum for 10 min did not activate Na+/H+ exchange; however, readdition of serum for 4 h restored Na+/H+ exchange to control values. This long-term effect of serum on Na+/H+ exchange activity could not be explained by changes in intracellular buffering capacity or intracellular [Na+]. 4-h serum deprivation reduced the K(t) of the exchanger for external Na+ from 21 to 6 mM, and reduced the V(max) by 57%, but did not alter the IC50 for amiloride in the presence of 140 mM Na+. Inhibition of protein synthesis with cycloheximide (5 microM) did not alter the effect of serum removal or readdition on Na+/H+ exchange. Low temperature (13 degrees C) completely prevented the inhibition of Na+/H+ exchange caused by the removal of serum. In addition, once Na+/H+ exchange was inhibited by serum removal at 37 degrees C, maintaining cells at 13 degrees C also blocked the recovery of Na+/H+ exchange caused by serum readdition. Conversely, cytochalasin D (0.1-20 microM) blocked the reduction of Na+/H+ exchange which occurred due to 4-h serum deprivation, but did not block the restoration of Na+/H+ exchange when the cells were re-exposed to serum for a further 4 h. Colchicine (20 microM) did not alter the effect of serum removal or readdition. These data suggest that serum regulates Na+/H+ exchange activity by a posttranslational mechanism which is dependent on F-actin.     

[3H]ethylpropylamiloride, a radio-labelled diuretic for the analysis of the Na+/H+ exchange system. Its use with kidney cell membranes.

The interaction of amiloride and several amiloride derivatives with the Na+/H+ exchange system in Madin-Darby canine kidney cells and in rabbit renal microvillus membrane vesicles was studied from 22Na+ uptake experiments. On both types of preparation, the order of potency of the different molecules tested is: ethylisopropylamiloride greater than ethylpropylamiloride (EPA) greater than amiloride greater than benzamil. 3H-labelled EPA was prepared and used to titrate amiloride binding sites in solubilized microvillus membranes. Kinetics experiments, equilibrium binding studies and competition experiments between [3H]EPA and unlabelled EPA indicate that EPA recognizes a single family of binding sites with a Kd value of 45 nM and a maximum binding capacity of 2 pmol/mg of protein. The order of potency of different amiloride analogs tested in [3H]EPA competition experiments is identical to that found for the inhibition of 22Na+ uptake by the Na+/H+ exchange system, suggesting that [3H]EPA binding sites are associated with the Na+/H+ exchange system. [3H]EPA binding sites are pharmacologically distinct from those of [3H]benzamil and [3H]bumetanide in kidney membranes.     

Analysis by base exchange of thyrotropin-releasing hormone responsive and unresponsive inositol lipid pools in rat pituitary tumor cells

We have shown that there is an inositol (Ins) lipid pool in cloned rat pituitary tumor (GH3) cells that is hydrolyzed in response to thyrotropin-releasing hormone (TRH) and an unresponsive pool. Because others have suggested that incorporation of [3H]Ins by base exchange may not occur uniformly into Ins lipids in other cell types, we established conditions using permeabilized cells under which labeling occurs by Ins-phosphatidylinositol (PI) exchange in the absence of de novo PI synthesis to further characterize these pools in GH3 cells. In permeabilized cells incubated in buffer containing 10 mM Mg2+ and 0.1 mM CMP, [3H]Ins incorporation into lipids occurred by base exchange only. This was so because: 1) [3H]Ins incorporation into lipids displayed properties similar to that for release of 3H-labeled Ins by unlabeled Ins from PI in cells prelabeled in situ prior to permeabilization; and 2) there was no change in PI mass under these conditions. In permeabilized cells incubated in buffer with 0.1 mM [3H]Ins for 60 min, incorporation was 0.61 +/- 0.05 nmol of [3H]Ins/10(6) permeabilized cells, which amounted to 35% of PI, while the level of PI, measured as nonradioactive phosphorus, was 94 +/- 8.0% of control. Permeabilized GH3 cells were responsive to TRH. In cells prelabeled in situ and then permeabilized, TRH stimulated an increase in 3H-labeled Ins phosphates (IPs) in 20 min which was 10% of 3H radioactivity initially present in lipids. This increase in 3H-labeled IPs was 6.3 times the 3H radioactivity present in phosphatidylinositol 4,5-bisphosphate prior to stimulation. When prelabeled cells were exchanged with unlabeled Ins after permeabilization there was only a 10-16% decrease in 3H-labeled IP accumulation stimulated by TRH even though 3H-labeled lipids decreased to 52% of control. TRH did not affect labeling by [3H]Ins-PI exchange. In cells labeled by base exchange after permeabilization TRH stimulated a very small increase in 3H-labeled IPs of only 0.21 +/- 0.02% of 3H-labeled lipids in 20 min or only 7% of the 3H radioactivity in phosphatidylinositol 4,5-bisphosphate. These data show that in permeabilized GH3 cells base exchange can occur in the absence of de novo PI synthesis and that lipids that are preferentially labeled by base exchange comprise a pool that is less responsive to TRH than total Ins lipids.     

Regulation of amino acid uptake by phorbol esters and hypertonic solutions in rat thymocytes

Growth factors, mitogens, and malignant transformation can alter the rate of amino acid uptake in mammalian cells. It has been suggested that the effects of these stimuli on proliferation are mediated by activation of Na+/H+ exchange. In lymphocytes, Na+/H+ exchange can also be activated by phorbol esters and by hypertonic media. To determine the relationship between the cation antiport and amino acid transport, we tested the effects of these agents on the uptake of alpha-aminoisobutyric acid (AIB), methyl-AIB, proline, and leucine in rat thymocytes. Both 12-O-tetradecanoylphorbol-13-acetate (TPA) and hypertonicity stimulated amino acid uptake through system A (AIB, proline, and methyl-AIB). In addition, TPA, but not hypertonicity, also elevated leucine uptake. The stimulation of the Na+ -dependent system A was not due to an increased inward electrochemical Na+ gradient. The effects of TPA and hypertonic treatment were not identical: Stimulation of AIB uptake by TPA was observed within minutes, whereas at least 1 hr was required for the effect of hypertonicity to become noticeable. Moreover, stimulation by hypertonicity but not that by TPA, was partially inhibited by cycloheximide, suggesting a role of protein synthesis. That stimulation of Na+/H+ exchange does not mediate the effects on amino acid transport is suggested by two findings: 1) the stimulation of AIB uptake was not prevented by concentrations of amiloride or of 5-(N,N-disubstituted) amiloride analogs that completely inhibit the Na+/H+ antiport and 2) conditions that mimic the effect of the antiport, namely, increasing [Na+]i or raising pHi failed to stimulate amino acid uptake. Thus, in lymphocytes, activation of Na+/H+ exchange and stimulation of amino acid transport are not casually related.     

Possible involvement of a Na+/H+ antiporter in the stimulation of hexose transport in Swiss 3T3 cells by a phorbol ester and growth factors

Phorbol-12,13-dibutyrate, epidermal growth factor, and insulin raised the intracellular pH ([pH]i), presumably through the activation of a Na+/H+ antiporter. Addition of amiloride or replacement of extra-cellular Na+ by choline which abolishes the cytoplasmic alkalinization prevented the stimulation of hexose transport by these agents. Furthermore, monensin, a Na+/H+ ionophore which increases the [pH]i, stimulated hexose transport. This stimulation was also prevented by the replacement of extra-cellular Na+ by choline. These observations suggest that stimulation of the Na+/H+ antiporter may have stimulated the increase in hexose transport.