Regulation of human neutrophil chemotaxis by intracellular pH

The relationship of N-formyl-methionyl-leucyl-phenylalanine-stimulated Na+/H+ exchange to the chemotactic responsiveness of human neutrophils was investigated. The pHi changes, measured from the equilibrium distribution of 5,5-dimethyloxazolidine-2,4-dione, were correlated with the migratory behavior of the cells as assessed by the leading front method. Exposure of cells to 10 nM FMLP caused activation of Na+/H+ exchange, leading to a rise in pHi from approximately 7.25 to approximately 7.75. This intracellular alkalinization was inhibited by amiloride and by three more potent analogues. All four compounds reduced the chemotactic response to FMLP with apparent Ki values similar to those for inhibition of the pHi transients, thereby suggesting that the blocking effect of the drugs on directed cell migration was related to inhibition of Na+/H+ exchange. The effect was specific for stimulated cell locomotion: FMLP-induced chemotaxis and chemokinesis were inhibited in parallel, whereas random motility was unimpaired. The relationship of pHi to function was also studied as the pHi of FMLP-activated cells was varied between 6.8 and 8.6 by altering the chemical gradients for Na+ and H+ across the cell membrane. There was a direct, positive correlation between the pHi value attained following FMLP-stimulation and the locomotor response to a chemotactic gradient. These results indicate that the motile functions of human neutrophils can be regulated by their pHi.     

Regulation of intracellular pH

The approach that most animal cells employ to regulate intracellular pH (pH(i)) is not too different conceptually from the way a sophisticated system might regulate the temperature of a house. Just as the heat capacity (C) of a house minimizes sudden temperature (T) shifts caused by acute cold and heat loads, the buffering power (beta) of a cell minimizes sudden pH(i) shifts caused by acute acid and alkali loads. However, increasing C (or beta) only minimizes T (or pH(i)) changes; it does not eliminate the changes, return T (or pH(i)) to normal, or shift steady-state T (or pH(i)). Whereas a house may have a furnace to raise T, a cell generally has more than one acid-extruding transporter (which exports acid and/or imports alkali) to raise pH(i). Whereas an air conditioner lowers T, a cell generally has more than one acid-loading transporter to lower pH(i). Just as a house might respond to graded decreases (or increases) in T by producing graded increases in heat (or cold) output, cells respond to graded decreases (or increases) in pH(i) with graded increases (or decreases) in acid-extrusion (or acid-loading) rate. Steady-state T (or pH(i)) can change only in response to a change in chronic cold (or acid) loading or chronic heat (or alkali) loading as produced, for example, by a change in environmental T (or pH) or a change in the kinetics of the furnace (or acid extrudes) or air conditioner (or acid loaders). Finally, just as a temperature-control system might benefit from environmental sensors that provide clues about cold and heat loading, at least some cells seem to have extracellular CO(2) or extracellular HCO(3)(-) sensors that modulate acid-base transport.     

Pertussis toxin inhibits intracellular pH changes in human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine

Changes of intracellular pH in human neutrophils were monitored by 9-aminoacridine fluorescence. Both initial acidification and subsequent alkalinization phases induced by a chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine were dependent on the extracellular Ca2+-concentrations, and a calcium ionophore, A-23187 similarly induced the pH-changes. Pertussis toxin inhibited the pH-changes induced by the peptide while cholera toxin did not. The pH-changes induced by A-23187 were not affected by the toxins. The results suggest that the inhibitory guanine-nucleotide regulatory protein and Ca2+ are involved in the pH-changes induced by the peptide.     

Receptor-mediated actin assembly in electropermeabilized neutrophils: role of intracellular pH

Neutrophil activation by a variety of stimuli is accompanied by an intracellular acidification, which has been postulated to mediate actin polymerization (Yuli and Oplatka, Science 1987, 235, 340). This hypothesis was tested using 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin staining and flow cytometry, or right angle light scattering to study actin assembly in intact and electrically permeabilized human neutrophils. Intracellular pH was measured fluorimetrically using a pH sensitive dye. In cells stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) at 21 degrees C, actin assembly clearly preceded the intracellular acidification in response to fMLP. Moreover, actin polymerization persisted in cells where intracellular pH was clamped near the resting (unstimulated) level using nigericin/K+. Finally, fMLP induced a significant increase in F-actin content in electropermeabilized neutrophils equilibrated with an extracellular medium containing up to 50 mM HEPES. These observations indicate that fMLP-stimulated F-actin assembly is not mediated by a decrease in intracellular pH and suggest that changes in transmembrane potential and ionic gradients are unlikely to mediate actin polymerization.     

Regulation of podosomes by intracellular pH in avian osteoclasts

The effects of changes in intracellular pH (pH1) on the organization of the clear zone of isolated avian osteoclasts in culture were studied. The distribution of podosomes, the close contact areas that mediate the adhesion of osteoclasts to the substrate, was investigated by decoration of microfilaments with fluorescent phalloidin. Intracellular acidification by butyric acid induces significant increase of podosome formation at the level of the clear zone compared to controls. Conversely, alkalinization by HCO3- reduces the percentage of osteoclasts with podosomes. A role of pH1 on the adhesion of the osteoclasts to the substrate is hypothesized.     

Altered intracellular pH regulation in neutrophils from patients with cystic fibrosis

Cystic fibrosis (CF) is a condition characterized by neutrophil-mediated lung damage and bacterial colonization. The physiological basis for reported functional alterations in CF neutrophils, including increased release of neutrophil elastase, myeloperoxidase, and oxidants, is unknown. These processes are, however, regulated by intracellular pH (pH(i)). We demonstrate here that pH(i) regulation is altered in neutrophils from CF patients. Although resting pH(i) is similar, pH(i) after acid loading and activation (N-formyl-methionyl-leucyl-phenylalanine and phorbol 12-myristate 13-acetate) is more acidic in CF cells than in normal cells. Furthermore, patients with non-CF-related bronchiectasis handle acid loading and activation in a fashion similar to subjects with normal neutrophils, suggesting that chronic pulmonary inflammation alone does not explain the difference in pH(i). This is further supported by data showing that normal neutrophils exposed to the CF pulmonary milieu respond by increasing pH(i) as opposed to decreasing pH(i) as seen in activated CF neutrophils. These pH(i) differences in activated or acid-loaded CF neutrophils are abrogated by ZnCl(2) but not by amiloride and bafilomycin A(1), suggesting that passive proton conductance is abnormal in CF. In addition, DIDS, which inhibits HCO(3)(-)/Cl(-) exchange, causes alkalinization of control but not of CF neutrophils, suggesting that anion transport is also abnormal in CF neutrophils. In summary, we have shown that pH(i) regulation in CF neutrophils is intrinsically abnormal, potentially contributing to the pulmonary manifestations of the condition.     

Regulation of intracellular pH in capacitated human spermatozoa by a Na+/H+ exchanger

We previously demonstrated that the progesterone‐ (P) initiated human sperm acrosome reaction (AR) was dependent on the presence of extracellular Na⁺ (Na⁺_o). Moreover, Na⁺_o depletion resulted in a decreased cytosolic pH (pH_i), suggesting involvement of a Na⁺‐dependent pH_i regulatory mechanism during the P‐initiated AR. We now report that the decreased pH_i resulting from Na⁺_o depletion is reversible and mediated by a Na⁺/H⁺ exchange (NHE) mechanism. To determine the role of an NHE in the regulation of pH_i, capacitated spermatozoa were incubated in Na⁺‐deficient, bicarbonate/CO₂‐buffered (0NaB) medium for 15–30 min, which resulted in an intracellular acidification as previously reported. These spermatozoa were then transferred to Na⁺‐containing, bicarbonate/CO₂‐buffered (NaB) medium; Na⁺‐containing, Hepes‐buffered (NaH) medium; or maintained in the 0NaB medium. Included in the NaH medium was the NHE inhibitor 5‐(N‐ethyl‐N‐isopropyl) amiloride (EIPA). The steady‐state pH_i was then determined by spectrofluorometric measurement of bis(carboxyethyl)‐5(6)‐carboxyfluoroscein (BCECF) fluorescence. EIPA (0.1 μM) significantly (P < 0.05) inhibited the pH_i recovery produced by NaH medium. Moreover, the pH_i in NaH medium was not significantly (P < 0.05) different than NaB medium. These results indicate that a Na⁺‐dependent, bicarbonate‐independent pH_i regulatory mechanism, with a pharmacological characteristic consistent with an NHE, is present in capacitated spermatozoa. In support of the involvement of a sperm NHE, we also demonstrated specific immunoreactivity for a 100 kDa porcine sperm protein using an NHE‐1 specific monoclonal antibody. Interestingly, no significant (P = 0.79) effect was seen on the P‐initiated AR when EIPA was included in either the NaH or NaB medium. While these findings suggest that inhibition of NHE‐dependent pH_i regulation in capacitated spermatozoa is not sufficient to block initiation of the AR by P, they do not preclude the possibility that an NHE mediates the regulation of capacitation or sperm motility. Mol. Reprod. Dev. 52:189–195, 1999. © 1999 Wiley‐Liss, Inc.     

Regulation of leucine transport by intracellular pH in Bacillus pasteurii

The kinetics, specificity and mechanism of leucine uptake were studied in the alkaliphilic bacterium Bacillus pasteurii DSM 33 (ATCC 11859). Leucine was accumulated up to 200-fold by a sodium-dependent secondary transport system for branched-chain amino acids. Apparent K_t values of 9.6 μM for leucine, 8.9 μM for isoleucine, 9.3 μM for valine, and 0.71 mM for sodium were determined, and maximum uptake activity was observed at an external pH of 8.5 and at 35°C. The effect of several ionophores indicated that transport was energized by the membrane potential and a sodium gradient; each gradient alone was sufficient to drive the uptake of leucine. The activity of the leucine transport system was regulated by the intracellular pH and was inhibited at an internal pH below 7.0. Received: 26 September 1995 / Accepted: 10 December 1995     

Specific modulation of the intracellular pH of rabbit neutrophils by chemotactic factors

We have monitored the intracellular pH of rabbit neutrophils by following the distribution of the weak acid, 5,5-Dimethyloxazolidine-2, 4-dione. The synthetic formyl-methionyl chemotactic factors were found to induce complex and specific changes in the intracellular pH of the neutrophils, a rapid drop followed by a slower and more sustained rise. These effects are receptor mediated. The relationship of these events to the physiology of the neutrophils is discussed.     

Intracellular pH regulation during spreading of human neutrophils

The regulation of the intracelluar pH (pHi) during spreading of human neutrophils was studied by a combination of fluorescence imaging and video microscopy. Spreading on adhesive substrates caused a rapid and sustained cytosolic alkalinization. This pHi increase was prevented by the omission of external Na+, suggesting that it results from the activation of Na+/H+ exchange. Spreading-induced alkalinization was also precluded by the compound HOE 694 at concentrations that selectively block the NHE-1 isoform of the Na+H+ antiporter. Inhibition of Na+/H+ exchange by either procedure unmasked a sizable cytosolic acidification upon spreading, indicative of intracellular acid production. The excess acid generation was caused, at least in part, by the activation of the respiratory burst, since the acidification closely correlated with superoxide production, measured in single spreading neutrophils with dihydrorhodamine-123, and little acid production was observed in the presence of diphenylene iodonium, a blocker of the NADPH oxidase. Moreover, neutrophils from chronic granulomatous disease patients, which do not produce superoxide, failed to acidify. Comparable pHi changes were observed when beta 2 integrins were selectively activated during spreading on surfaces coated with anti-CD18 antibodies. When integrin engagement was precluded by pretreatment with soluble anti-CD18 antibody, the pHi changes associated with spreading on fibrinogen were markedly reduced. Inhibition of microfilament assembly with cytochalasin D precluded spreading and concomitantly abolished superoxide production and the associated pHi changes, indicating that cytoskeletal reorganization and/or an increase in the number of adherence receptors engaged are required for the responses. Neutrophils spread normally when the oxidase was blocked or when pHi was clamped near physiological values with nigericin. Spreading, however, was strongly inhibited when pHi was clamped at acidic values. Our results indicate that neutrophils release superoxide upon spreading, generating a burst of intracellular acid production. The concomitant activation of the Na+/H+ antiport not only prevents the deleterious effects of the acid released by the NADPH oxidase, but induces a net cytosolic alkalinization. Since several functions of neutrophils are inhibited at an acidic pHi, the coordinated activation of pHi regulatory mechanisms along with the oxidase is essential for sustained microbicidal activity.     

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.     

Regulation of intracellular pH in the myocardium; relevance to pathology

Intracellular pH affects the contractile function of the heart, metabolic reactions, ion exchange and calcium homoeostasis. Numerous studies have concluded that a fall of extracellular pH, by whatever mechanism, causes a fall of contractility by alteration of intracellular pH. Measurement of cytosolic intracellular pH using microelectrodes has confirmed that earlier deduction. Acidosis reduces the slow calcium current and the release of calcium from the sarcoplasmic reticumul but, because the cytosolic calcium does not fall, the major site of action of hydrogen ions appears to be on the calcium sensitivity of the contractile proteins. In man acidosis can be detected 15 s after the occlusion of a coronary artery and is a major mechanism for the simultaneous loss of contractility in ischaemia. A transient alkalosis is not detected in man but has been reported in isolated heart preparations where ATP consumption is low.An imposed mild respiratory acidosis during hypoxia increases the subsequent recovery of mechanical function on reoxygenation whereas a severe acidosis can be harmful. Acidosis in ischaemic may be advantageous due to a cardioplegic effect, inhibition of transsarcolemmal calcium fluxes or a reduction of mitochondrial calcium overload. Calcium uptake on reperfusion or reoxygenation has been linked to an inward movement of sodium in exchange for hydrogen ions on reperfusion and subsequent sodium-calcium exchange. Such a mechanism in its simplest form cannot account for the similar uptake of calcium on reoxygenation and reperfusion. Acidosis is a cause of early contractile failure in ischaemia but the role of acidosis in causing cell necrosis is not established.