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.     

Binding of3H-phenamil,an irreversible amiloride analog,to toad urinary bladder: effects of aldosterone and vasopressin

Summary Phenamil, an analog of amiloride, has previously been shown to bind specifically to sodium channels in toad bladder (J.L. Garvin et al.,J. Membrane Biol. 87:45–54, 1985). In this paper,³H-phenamil was used to measure sodium channel density in both isolated epithelial cells and intact bladders. From the specific binding to intact bladders, a channel density of 455±102 channels/m² was calculated. No correlation between specific binding and the magnitude of irreversible inhibition of shortcircuit current was found. Pretreatment of intact bladders with 1 mg/ml trypsin reduced specific binding to isolated cells by 82±5%. In isolated cells, neither aldosterone nor vasopressin had any significant effect on specific phenamil binding. It is inferred that phenamil binds to both open and closed channels which may be either in the mucosal membrane or in the submembrane space. Finally, and rather surprisingly, we found that³H-phenamil binds irreversibly to the basolateral membrane at concentrations as low as 4×10^–7 m. Therefore, care must be used in interpreting binding studies with amiloride or its analog at such concentrations.     

Identification of a common signal associated with cellular proliferation stimulated by four haemopoietic growth factors in a highly enriched population of granulocyte/macrophage colony-forming cells.

We have prepared a population of bone marrow cells that is highly enriched in neutrophil/macrophage progenitor cells (GM-CFC). Four distinct haemopoietic growth factors can stimulate the formation of mature cells from this population, although the proportions of neutrophils and/or macrophages produced varied depending on the growth factor employed: interleukin 3 (IL-3) and granulocyte/macrophage colony-stimulating factor (GM-CSF) stimulated the formation of colonies containing both neutrophils and macrophages; macrophage colony-stimulating factor (M-CSF) produced predominantly macrophage colonies; and granulocyte colony-stimulating factor (G-CSF) promoted neutrophil colony formation. Combinations of these four growth factors did not lead to any additive or synergistic effect on the number of colonies produced in clonal soft agar assays, indicating the presence of a common set of cells responsive to all four haemopoietic growth factors. These enriched progenitor cells therefore represent an ideal population to study myeloid growth-factor-stimulated survival, proliferation and development. Using this population we have examined the molecular signalling mechanisms associated with progenitor cell proliferation. We have shown that modulation of cyclic AMP levels has no apparent role in GM-CFC proliferation, whereas phorbol esters and/or Ca2+ ionophore can stimulate DNA synthesis, indicating a possible role for protein kinase C activation and increased cytosolic Ca2+ levels in the proliferation of these cells. The lack of ability of all four myeloid growth factors to mobilize intracellular Ca2+ infers that these effects are not achieved via inositol lipid hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and purification of a renal amiloride-binding protein with properties of the Na+-H+ exchanger

The aim of this study was to identify and purify the Na+-H+ exchanger from rabbit renal brush border membranes by use of affinity chromatography. Triton-solubilized membranes were equilibrated with an affinity matrix consisting of the amiloride analogue A35 (5-N-(3-aminophenyl)amiloride) covalently coupled to Sepharose CL-4B beads through a triglycine spacer arm. The matrix was then washed extensively with buffer and sequentially eluted with buffer, buffer containing 5 mM amiloride, and 1% sodium dodecyl sulfate (SDS). Eluates were concentrated and subjected to SDS-polyacrylamide gel electrophoresis. The silver-stained gel revealed a 25-kDa protein that was not visible in the initial solubilized brush border membrane extract, was not eluted from the affinity matrix by buffer alone, but was eluted with 5 mM amiloride. A subsequent elution with 1% SDS did not release any more of the 25-kDa protein, indicating that it had been completely eluted from the affinity matrix by amiloride. The presence of 5 mM amiloride during equilibration of the solubilized brush border extract with the affinity matrix completely blocked adsorption of the 25-kDa protein. The relative abundance of this protein correlated closely with Na+-H+ exchange activity when preparations of cortical brush border membrane vesicles, outer medullary brush border membrane vesicles, and cortical basolateral membrane vesicles were compared. Moreover, binding of the protein to the affinity matrix was inhibited by amiloride and amiloride analogues with a rank order identical to that for inhibition of Na+-H+ exchange activity. These findings strongly suggest that the 25-kDa protein is a structural component of the Na+-H+ exchanger.     

ATP-dependent and DCCD-insensitive Cl- uptake by membrane vesicles from the rat brain plasma membrane fractions

ATP-dependent Cl- uptake by membrane vesicles from the rat brain plasma membrane fractions was not affected by the addition of 40 mM of K+, Na+ or HCO3- to the assay medium. Na+ and K+ did not alter the uptake even in the presence of a K+ ionophore, valinomycin (10 microM), or a H+/K+ exchanger, nigericin (10 microM), whereas in the presence of both of these ionophores, K+, but not Na+, reduced the Cl- uptake. Inhibitors of proton pump activity, N,N'-dicyclohexylcarbodiimide (1 mM) and 5-(N,N-hexamethylene)amiloride (40 microM), however, did not affect the Cl- uptake. These findings suggest the presence of a primary Cl- transport system probably associated with passive H+ flux in the brain plasma membranes.     

Kinetic properties of the Na+/H+ antiport of heart mitochondria

The fluorescence of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) has been used to follow the Na+/H+ antiport activity of isolated heart mitochondria as a Na+-dependent extrusion of matrix H+. The antiport activity measured in this way shows a hyperbolic dependence on external Na+ or Li+ concentration when the external pH (pHo) is 7.2 or higher. The apparent Km for Na+ decreases with increasing pHo to a limit of 4.6 mM. The Ki for external H+ as a competitive inhibitor of Na+/H+ antiport averages 3.0 nM (pHo 8.6). The Vmax at 24 degrees C is 160 ng ion of H+ min-1 (mg of protein)-1 and does not vary with pHo. Li+ reacts with the antiporter with higher affinity, but much lower Vmax, and is a competitive inhibitor of Na+/H+ antiport. The rate of Na+/H+ antiport is optimal when the pHi is near 7.2. When pHo is maintained constant, Na+-dependent extrusion of matrix H+ shows a hyperbolic dependence on [H+]i with an apparent Km corresponding to a pHi of 6.8. The Na+/H+ antiport is inhibited by benzamil and by 5-N-substituted amiloride analogues with I50 values in the range from 50 to 100 microM. The pH profile for this inhibition seems consistent with the availability of a matrix binding site for the amiloride analogues. The mitochondrial Na+/H+ antiport resembles the antiport found in the plasma membrane of mammalian cells in that Na+, Li+, and external H+ appear to compete for a common external binding site and both exchanges are inhibited by amiloride analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Paradoxical effects of amiloride analogs on protein phosphorylation and serotonin release induced by agonists in human platelets

We examined the effects of newly exploited amiloride analogs on protein phosphorylation and serotonin secretion induced by various agonists in human platelets. 3', 4'-dichlorobenzamil (DCB) and to a lesser extent, 2', 4'-dimethylbenzamil (DMB), which in many cells highly specific inhibitors of Na+/Ca2+-pump, inhibited the phosphorylation of 47K- and 20K-dalton proteins and serotonin secretion in human platelets independently of the action on the pump. DCB also induced dephosphorylation of 47K and 20K after the phosphorylation of these proteins by thrombin and released serotonin by itself.     

Conductive sodium pathway with low affinity to amiloride in LLC-PK1 cells and other epithelia

Electrical potential driven 22Na+ fluxes were measured in membrane vesicles prepared from a number of cultured and naturally occurring epithelia. In all preparations a rheogenic pathway blocked by 200 microM (but not by 1.5 microM) amiloride was noted. This transporter was characterized in membranes prepared from cultured LLC-PK1 cells. In this preparation more than 50% of the rheogenic 22Na+ uptake was blocked by amiloride (IC50 approximately 30 microM), phenamil (IC50 approximately 66 microM), or ethylisopropylamiloride (IC50 approximately 5 microM). This amiloride-sensitive flux was not seen if the vesicles were partially depolarized by external Na+ or K+. It could not be driven by a pH gradient, did not require the presence of Ca2+, sugars, or amino acids, and showed little dependence on temperature (25 versus 0 degrees C). The data suggest the existence of an epithelial amiloride-blockable Na+ transporter different from the previously characterized Na+ channel, Na+/H+ and Na+/Ca2+ exchangers, and the Na+-hexose co-transporter. In rat kidney cortex membranes prepared by Mn2+ precipitation, this transporter is primarily located in the brush-border fraction.     

Effects of glucocorticoids on Na+/H+ exchange and growth in cultured vascular smooth muscle cells

We have examined the effects of hydrocortisone on growth and Na+/H+ exchange in cultured rat aortic vascular smooth muscle cells (VSMC). Hydrocortisone (2 microM) treatment of growth-arrested VSMC significantly decreased VSMC growth in response to 10% calf serum assayed by 3H-thymidine incorporation and cell number at confluence. This effect was associated with the appearance of an altered cell phenotype characterized by large, flat VSMC that did not form typical "hillocks." Na+/H+ exchange was also altered in hydrocortisone-treated cells assayed by dimethylamiloride-sensitive 22Na+ influx into acid-loaded cells or by intracellular pH (pHi) change using the fluorescent dye BCECF. Resting pHi was 7.25 +/- 0.04 and 7.15 +/- 0.05 in control and hydrocortisone-treated cells, respectively (0.1 less than P less than 0.05). Following intracellular acidification in the absence of external Na+, pHi recovery upon addition of Na+ was increased 89% in hydrocortisone-treated cells relative to control. This was due to an increase in the Vmax for the Na+/H+ exchanger from 17.5 +/- 2.4 to 25.9 +/- 2.0 nmol Na+/mg protein x min (P less than 0.01) without a significant change in Km. Treatment of VSMC with actinomycin D (1 microgram/ml) or cycloheximide (10 microM) completely inhibited the hydrocortisone-mediated increase in Na+/H+ exchange, indicating a requirement for both RNA and protein synthesis. Because hydrocortisone altered the Vmax for Na+/H+ exchange, in contrast to agonists such as serum or angiotensin II which alter the Km for intracellular H+ or extracellular Na+, respectively, we studied the effect of hydrocortisone on activation of Na+/H+ exchange by these agonists. In cells maintained at physiological pHi (7.2), the initial rate (2 min) of angiotensin II-stimulated alkalinization was increased 66 +/- 39% in hydrocortisone-treated compared with control cells. Hydrocortisone caused no change in angiotensin II-stimulated phospholipase C activity assayed by measurement of changes in intracellular Ca2+ or diacylglycerol formation. However, angiotensin II and serum stimulated only small increases in Na+/H+ exchange in acid-loaded (pHi = 6.8) hydrocortisone-treated cells. These findings suggest that hydrocortisone-mediated increases in VSMC Na+/H+ exchange occur in association with a nonproliferating phenotype that has altered regulation of Na+/H+ exchange activation. We propose that hydrocortisone-mediated growth inhibition may be a useful model for studying the role of Na+/H+ exchange in cell growth responsiveness.     

A Na(+)-dependent Ca2+ exchanger generates the sustained increase in intracellular Ca2+ required for T cell activation.

Movement of extracellular Ca2+ is required for the sustained increase in [Ca2+]i necessary for T cell activation. However, the mechanisms mediating mitogen-stimulated Ca2+ movement into T cells have not been completely delineated. To explore the possibility that a Na(+)-dependent Ca2+ (Na+/Ca2+) exchanger might play a role in the mitogen-induced increases in [Ca2+]i required for T cell activation, the effects of inhibitors of this exchanger were examined. Inhibitors of Na+/Ca2+ exchange suppressed the sustained increase in [Ca2+]i stimulated by ligation of the CD3-TCR complex, but did not affect mobilization of intracellular Ca2+ stores. Consistent with the importance of this prolonged increase in [Ca2+]i in T cell activation, Na+/Ca2+ exchange inhibitors, but not inhibitors of the Na+/H+ antiporter, inhibited DNA synthesis stimulated by immobilized anti-CD3 mAb. Inhibition only occurred when the agents were present during the first hours after stimulation. These agents also inhibited IL-2 production, but not expression of the IL-2R or of an early activation Ag, 4F2. Inhibition of IL-2 production did not account for the inhibition of T cell proliferation as addition of exogenous IL-2 or phorbol ester (PDB) did not overcome the inhibition. In contrast, activation pathways that are not thought to require an increase in [Ca2+]i such as IL-1 + PDB or engagement of CD28 in the presence of PDB were less sensitive to the suppressive effects of inhibitors of Na+/Ca2+ exchange. Thus, proliferation induced by these stimuli was not suppressed by low concentrations of these inhibitors and IL-2 production induced by mAb to CD28 + PDB was not inhibited by any concentration of inhibitors of Na+/Ca2+ exchange. These results suggest that stimulation of a Ca2+ transporter with the same spectrum of inhibition as the Na+/Ca2+ exchanger in other tissues mediates the sustained increase in [Ca2+]i required for T cell activation after CD3 ligation.