Ion transport, membrane potential, and cytoplasmic pH in lymphocytes: changes during activation.
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Animals↗
Biomedical subjects
S Grinstein
Publications and source records attributed to S Grinstein.
Calcium homeostasis and the activation of calcium channels in cells of the immune system.
In summary, calcium seems to play a central role in the activation of cells of the immune system. When the cells are stimulated, [Ca2+]i generally increases as a result of entry from the external medium, as well as mobilization of calcium from intracellular membrane-bound compartments. It is generally acknowledged that inositol 1,4,5-trisphosphate mediates the release of calcium from internal stores. By comparison, relatively little is known about the mechanism underlying entry of calcium across the plasma membrane. Currently available evidence seems to rule out the participation of classic voltage-gated channels. Similarly, it appears unlikely that the interaction of ligands with their surface receptors directly activates the channels. Instead, it is more likely that a second messenger produced by the formation of the receptor-ligand complex is responsible for gating the channels. Several mediators, including calcium itself and inositol phosphates, have been proposed to effect gating of the channels, but conclusive evidence is not yet available. Ongoing work is likely to reveal the nature of the putative second messenger(s) in the near future.
Calcium↗
Cytoplasmic pH regulation in macrophages by an ATP-dependent and N,N'-dicyclohexylcarbodiimide-sensitive mechanism. Possible involvement of a plasma membrane proton pump.
Cytoplasmic pH (pHi) regulation was studied in thioglycolate-elicited murine macrophages using fluorescent probes. Acid-loaded macrophages regained normal pHi by extrusion of H+ equivalents across the plasma membrane. A fraction of this recovery was due to Na+/H+ exchange, as evidenced by its partial Na+ dependence and amiloride sensitivity. The residual, Na+-independent pHi recovery (approximately 50% of the total) persisted in the nominal absence of HCO3- and was insensitive to disulfonic stilbenes, ruling out mediation by anion exchange. In contrast, intracellular alkalinization and H+ extrusion from the cells were inhibited by N-ethylmaleimide, by N,N'-dicyclohexylcarbodiimide or by prior depletion of intracellular ATP. These observations are consistent with the existence of a H+-pumping ATPase in the plasma membrane of macrophages. The mechanism of activation of the ATP-dependent H+ extrusion process was also investigated. In other systems, Ca2+ mobilization has been suggested to signal an exocytic insertion of H+ pumps into the plasma membrane. Acid loading of macrophages was accompanied by an elevation of the cytosolic Ca2+ concentration ([Ca2+]i), measured using indo-1. These results are consistent with a role for Ca2+ mobilization in the activation of H+ extrusion.
Adenosine Triphosphate↗
Interleukin-2 induces proliferation of T lymphocyte mutants lacking protein kinase C.
We have identified a murine T lymphocyte clone that apparently lacks diacylglycerol- and phospholipid-activated protein kinase C (PKC): cell extracts do not display phosphatidylserine, Ca2+, or phorbol ester-dependent phosphotransferase activity; the enzyme was not detected in immunoblots with PKC-specific antibodies; phorbol ester binding sites are not detectable in intact cells; and activators of PKC do not stimulate proliferation or Na+/H+ exchange in intact cells. Only PKC beta mRNA was detected in normal murine T lymphocytes. The mutant T lymphocytes contained amounts of 4.4 kb PKC beta message similar to those in normal murine lymphocytes, but the 2.9 kb and 1.2 kb messages found in normal lymphocytes were barely detectable. No abnormalities were detected on Southern analysis, suggesting that the abnormality may be at the level of message splicing or stability. Since PKC-deficient cells proliferate in response to the T lymphocyte growth factor, interleukin-2, we conclude that activation of PKC is not essential for the growth-promoting action of interleukin-2.
Animals↗
Mitogenic and non-mitogenic ligands trigger a calcium-dependent cytosolic acidification in human T lymphocytes.
We have investigated the patterns of cytosolic pH and Ca2+ ([Ca2+]i) changes after exposure of human peripheral blood T cells to different mitogenic and non-mitogenic ligands. Using ligands that have different accessory cell requirements and varying effect on [Ca2+]i or cell proliferation, we observed that intracellular acidification occurred only with agents that increased [Ca2+]i. However, treatment of the cells with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, results in significant cytosolic alkalinization without detectable acidification, but did not affect the proliferative responses to mitogenic ligands and was a potent co-mitogen with non-mitogenic ligands. These data indicate that initial acidification or alkalinization responses are not essential for early activation or triggering of DNA synthesis.
Antibodies, Monoclonal↗
Detection of ligand-activated conductive Ca2+ channels in human B lymphocytes.
It has been assumed that uptake of extracellular Ca2+ occurs through ligand-activated Ca2+ channels in anti-IgM stimulated human B cells. If so, then uptake should be associated with a depolarizing inward current. Instead, a hyperpolarization due to Ca2+-sensitive K+ conductance is observed. To demonstrate conductive Ca2+ channels in human B lymphocytes, we loaded the cells with 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetate (BAPTA), an intracellular Ca2+ chelating agent. This increased the magnitude of the Ca2+ current and delayed the Ca2+-dependent K+ conductance. In BAPTA-loaded B cells suspended in Ca2+-free medium and activated with anti-IgM, reintroduction of Ca2+ resulted in a depolarization that was inhibited by high (microM) concentrations of verapamil and was observed when Ca2+ was replaced by Ba2+ but not Mg2+. These data demonstrate the opening of selective, Ca2+ conductive channels in human B cells following cross-linking of surface immunoglobulins.
B-Lymphocytes↗
Activation of Na+/H+ exchange and the expression of cellular proto-oncogenes in mitogen- and phorbol ester-treated lymphocytes.
It has been suggested that an intracellular alkalinization, resulting from stimulation of Na+/H+ exchange, is a necessary step and perhaps the signal leading to cellular proliferation in cells stimulated by mitogens. This hypothesis was tested by measuring the early stages of the proliferative cascade in cells where antiport activity was precluded by omission of Na+ or by the addition of potent amiloride analogs. To circumvent possible nonspecific effects due to long incubations under these conditions, an early response to mitogens, the increased level of c-fos mRNA, was monitored. In rat thymic lymphocytes, the increase in the level of c-fos RNA induced by the combination of 12-O-tetradecanoylphorbol 13-acetate and ionomycin was unaffected by inhibition of the antiport with 5-(N-ethyl-N-propyl)amiloride. Increased c-fos RNA was also observed in the absence of Na+ and when alkalinization was prevented by means of nigericin. Similar results were obtained with phytohemagglutinin-stimulated human T lymphocytes. Moreover, although the lectin stimulated the antiport in these cells, an alkalinization was not observed, due to the concomitant occurrence of an acidifying process. It was concluded that the stimulation of the Na+/H+ antiport that accompanies the addition of mitogens is neither sufficient nor necessary for the initiation of cellular proliferation.
Amiloride↗
Receptor-mediated activation of electropermeabilized neutrophils. Evidence for a Ca2+- and protein kinase C-independent signaling pathway.
Electrically permeabilized neutrophils were used to study the mechanism of activation of the respiratory burst by the chemotactic agent formyl-methionyl-leucyl-phenylalanine (fMLP). Permeabilization was assessed by flow cytometry, radioisotope trapping, and by the requirement for exogenous NADPH for oxygen consumption. A respiratory burst could be elicited by fMLP, phorbol ester, or diacylglycerol in permeabilized cells suspended in EGTA-buffered medium with 100 nM free Ca2+. The fMLP response persisted even in cells depleted of intracellular Ca2+ stores by pretreatment with ionomycin. Therefore, a change in cytosolic free Ca2+ ([Ca2+]i) is not required for receptor-mediated stimulation of the respiratory burst. The responses induced by phorbol ester and diacylglycerol were largely inhibited by H7, a protein kinase C antagonist. In contrast, the stimulation of oxygen consumption by fMLP was unaffected by H7. These results suggest that a third signaling pathway, distinct from changes in [Ca2+]i and activation of protein kinase C, is involved in the response of neutrophils to chemoattractants.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Calcium-dependent intracellular acidification dominates the pH response to mitogen in human T cells.
The effects of a phorol ester and a mitogenic lectin on the intracellular pH (pHi) of human T lymphocytes was investigated. In contrast to the cytoplasmic alkalinization induced by 12-0-tetradecanoylphorbol-13-acetate, an acidification was recorded in cells treated with phytohemagglutinin. This decrease in pHi was magnified in Na+-free medium or in the presence of amiloride analogues, suggesting that activation of Na+/H+ exchange partially counteracts the phytohemagglutinin-induced acidification. The decrease in pHi was dependent on a sustained increase in cytosolic free Ca2+ and could be mimicked by addition of the divalent cation ionophore, ionomycin. The elevation of cytosolic free Ca2+ leads to metabolic H+ (equivalent) generation with consequent cytoplasmic acidification, which in human T cells predominates over the concurrent activation of the Na+/H+ antiport. These findings argue against the notion that activation of Na+/H+ exchange is a signal for the initiation of proliferation.
Calcium↗
Differential role of cation and anion exchange in lymphocyte pH regulation.
In lymphocytes, the Na+/H+ antiport is well suited to function in cytoplasmic pH (pHi) regulation. It is activated by departures from the physiological pHi and is thermodynamically poised to compensate for the tendency of the cells to become acidic. The driving force for H+ (equivalent) efflux is indirectly provided by the Na+ pump. Lymphocytes also possess a cation-independent anion (Cl-/HCO3-) exchange system, which, under the appropriate conditions, tends to restore pHi after an alkali load. Unlike the cation antiport, the source of energy driving the anion exchanger, i.e. the factors that determine the transmembrane Cl- distribution, is not well understood. The contribution of conductive pathways appears to be minimal, resulting in a marked difference between the membrane potential and ECl-. Instead, ECl- is very similar to EH+. Moreover, changes in the distribution of Cl- lead to alterations in the transmembrane delta pH and vice versa, suggesting a relationship between these parameters. Evidence is presented which suggests that the transmembrane distribution of HCO3-, dictated by delta pH, is a major determinant of the intracellular Cl- concentration, a process mediated by the anion exchanger. Thus, if Cl- is driven by the gradient of HCO3-, the cation-independent anion exchanger cannot play an active role in determining pHi. Instead, Cl-/HCO3- exchange may simply stabilize pHi by increasing the dynamic buffering power of the cells. Cation-independent Cl-/HCO3- exchange could be involved in pHi regulation only if coupled to a separate mechanism of intracellular Cl- accumulation, such as Na+-K+-2Cl- co-transport or an inward Cl- pump, which have not been detected in lymphoid cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Uptake of extracellular Ca2+ and not recruitment from internal stores is essential for T lymphocyte proliferation.
Changes in free cytosolic calcium concentrations ([Ca2+]i) are thought to be important initiating events in the activation of T lymphocytes. Mitogen-induced increases in [Ca2+]i may result from net influx across the plasma membrane and/or release of Ca2+ from intracellular stores. In human T lymphocytes loaded with the fluorescent indicator indo-1, addition of phytohemagglutinin (PHA) or the anti-CD3 antibody UCHT-1 elicits a biphasic [Ca2+]i response. A major component of the initial transient peak was due to release from internal stores whereas the lower plateau phase was sustained by Ca2+ influx. Previous work suggested that Ca2+ influx is essential for interleukin 2 (IL 2) secretion and cell proliferation. To determine the relative effects of the initial and sustained phases of [Ca2+]i change, IL 2 secretion and cell proliferation, we introduced into the cell 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a high affinity intracellular Ca2+ chelator which neither contributes to nor interferes with the fluorescence determinations of [Ca2+]i. In cells preloaded with BAPTA, both PHA and UCHT-1 antibody failed to elicit the transient [Ca2+]i overshoot. Only the plateau phase could be observed in the presence of extracellular Ca2+. In contrast, BAPTA-loaded cells were found to be fully functional when assessed for IL 2 receptor expression, IL 2 secretion and cell proliferation. Thus, the mitogen-induced, maximal but transient increase in [Ca2+]i, contributed to mainly by release of Ca2+ from internal stores, does not appear to be essential for these T cell responses.
Antibodies, Monoclonal↗
Cytosolic calcium, oxygen consumption and the intracellular pH of stimulated neutrophils.
The cytoplasmic pH undergoes a biphasic change when neutrophils are activated. The role of Ca2+ in initiating these changes was investigated. No correlation was found between the increased cytosolic [Ca2+] and the stimulation of the Na+/H+ antiport. Similarly, the cytoplasmic acidification elicited by activation in Na+-free media was found to be unrelated to [Ca2+]. Reversal of Na+/H+ exchange was also ruled out as the source of the acidification. Data using a variety of soluble activators indicate that metabolic acid generation is largely responsible for the observed drop in cytoplasmic pH.
Arachidonic Acid↗
Na+/H+ exchange and the regulation of intracellular pH in polymorphonuclear leukocytes.
1. Regulation of the cytoplasmic pH(pHi) was studied in quiescent and activated human neutrophils. Acid-loaded unstimulated cells regulate pHi by activating an electroneutral Na+/H+ exchange. 2. When activated, neutrophils undergo a biphasic change in pHi: an acidification followed by an alkalinization. The latter is due to stimulation of the Na+/H+ antiport. 3. The acidification, which is magnified in Na+-free or amiloride-containing media, is associated with net H+ efflux from the cells. 4. A good correlation exists between cytoplasmic acidification and superoxide generation: inhibition of the latter by adenosine, deoxyglucose or pertussis toxin also inhibits the pHi changes. 5. Moreover, acidification is absent in chronic granulomatous disease patients, which cannot generate superoxide. 6. Regulation of pHi is essential for neutrophil function. The oxygen dependent bactericidal activity is inhibited upon cytoplasmic acidification. This can result from impairment of Na+/H+ exchange, or from influx of exogenous acid equivalents. 7. The latter mechanism may account for the inability of neutrophils to resolve bacterial infections in abscesses, which are generally made acidic by accumulation of organic acids that are by-products of bacterial anaerobic metabolism.
Abscess↗
The Ayerst award lecture 1987/La Conférence Ayerst 1987. The intracellular pH of white blood cells: measurement and regulation.
This review summarizes recent progress in our understanding of the mechanisms of intracellular pH regulation, with particular emphasis on Na+-H+ countertransport. Methods developed for the measurement of cytoplasmic pH in small cells and their application to mammalian lymphocytes and neutrophils are described. The basic properties of the Na+-H+ antiport in these cells and its regulation by growth factors and tumor promoters will be discussed briefly. Finally, the significance of cytoplasmic pH regulation to cell function and survival will be considered.
Animals↗
Assessment of Na+-H+ exchange activity in phagosomal membranes of human neutrophils.
To assess the presence of Na+-H+ exchange in internalized membranes, the phagosomal pH was monitored in suspensions of intact human neutrophils by measuring the fluorescence of ingested bacteria (Micrococcus lysodeikticus) prelabeled with a pH-sensitive dye. Uptake of fluoresceinated bacteria was confirmed by flow cytometry and by phase-contrast and electron microscopy. Manipulation of the cytoplasmic ion content was accomplished by permeabilization of the plasma membrane with nystatin, which did not alter phagosomal permeability. At 37 degrees C, the phagosomal interior acidified at a maximal rate of 0.135 +/- 0.003 pH units/min (n = 10). The endogenous Na+-H+ exchanger does not affect phagosomal acidification, since the rate and extent of the pH change were not altered by 1) omission of intraphagosomal Na+ and 2) addition of the permeant inhibitor methylisobutylamiloride or by trapping amiloride in the phagosome during bacterial ingestion. Moreover, amiloride-sensitive Na+-H+ exchange was not detectable when Na+ or H+ gradients were imposed across the phagosomal membrane. Under comparable conditions, Na+-H+ exchange could be readily detected across the surface membrane. These data imply that the Na+-H+ antiporters are either inactivated in the phagosome or are segregated and not internalized into the phagosomal membrane.
Carrier Proteins↗
Activation of Ca2+-dependent K+ channels in human B lymphocytes by anti-immunoglobulin.
Many mammalian cell types exhibit Ca2+-dependent K+ channels, and activation of these channels by increasing intracellular calcium generally leads to a hyperpolarization of the plasma membrane. Their presence in B lymphocytes is as yet uncertain. Crosslinking Ig on the surface of B lymphocytes is known to increase the level of free cytoplasmic calcium ([Ca2+]i). However, rather than hyperpolarization, a depolarization has been reported to occur after treatment of B lymphocytes with anti-Ig. To determine if Ca2+-dependent K+ channels are present in B lymphocytes, and to examine the relationship between intracellular free calcium and membrane potential, we monitored [Ca2+]i by means of indo-1 and transmembrane potential using bis(1,3-diethylthiobarbituric)trimethine oxonol in human tonsillar B cells activated by anti-IgM. Treatment with anti-IgM induced a biphasic increase in [Ca2+]i and a simultaneous hyperpolarization. A similar hyperpolarization was induced by ionomycin, a Ca2+ ionophore. Delaying the development of the [Ca2+]i response by increasing the cytoplasmic Ca2+-buffering power delayed the hyperpolarization. Conversely, eliminating the sustained phase of the [Ca2+]i response by omission of external Ca2+ abolished the prolonged hyperpolarization. In fact, a sizable Na+-dependent depolarization was unmasked. This study demonstrates that in human B lymphocytes, Ca2+-dependent K+ channels can be activated by crosslinking of surface IgM. Moreover, it is likely that, by analogy with voltage-sensitive Ca2+ channels, Na+ can permeate through these ligand-gated Ca2+ "channels" in the absence of extracellular Ca2+.
Antibodies, Anti-Idiotypic↗
pH-dependent impairment of the neutrophil respiratory burst by the Bacteroides byproduct succinate.
The mechanisms by which Bacteroides species contribute to the virulence of polymicrobial intraabdominal infections are poorly defined. One potential mechanism may be their ability to inhibit host defenses, thereby permitting their co-pathogens to survive and exert their intrinsic virulence. The purpose of these studies was to examine the effect of the major Bacteroides byproduct, succinate, on the generation of the respiratory burst by neutrophils. Succinate caused a dose-dependent inhibition of both superoxide and hydrogen peroxide production. This phenomenon was markedly pH-dependent, i.e. inhibition occurred following incubation of cells in succinate at pH 5.5 but not at pH 7.4. It was further hypothesized that succinate effected this inhibition by reducing cytoplasmic pH. Presumably, at low pH, succinate in its protonated form traversed the plasma membrane and dissociated within the cytoplasmic space, thereby reducing pH. The cytoplasmic pH was determined fluorimetrically. Succinate-containing medium at pH 5.5 significantly reduced cytoplasmic pH compared to both control medium pH 5.5 and succinate medium pH 7.4. In further support of the hypothesis, it was shown that uptake of 14C-succinate by neutrophils was 8-fold increased at pH 5.5 compared to pH 7.4. Reduced temperature (4 degrees C) protected cells from the inhibitory effect of succinate at pH 5.5. Measurements of cytoplasmic pH and uptake of 14C-succinate suggested that this protection was achieved by retarding the uptake of succinic acid at low temperature. These studies suggest that fatty acid production by Bacteroides species in the acid milieux of an infection may be a potential mechanism by which Bacteroides species enhance the virulence of mixed infections.
Bacteroides↗
Intracellular pH regulation in neutrophils: properties and distribution of the Na+/H+ antiporter.
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Carrier Proteins↗