Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient

Two-photon fluorescence lifetime imaging is used to identify microdomains (1-25 microm) of two distinct pH values within the uppermost layer of the epidermis (stratum corneum). The fluorophore used is 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), whose lifetime tau (pH 4.5, tau = 2.75 ns; pH 8.5, tau = 3.90 ns) is pH dependent over the pH range of the stratum corneum (pH 4.5 to pH 7.2). Hairless mice (SKH1-hrBR) are used as a model for human skin. Images (< or =50 microm x 50 microm) are acquired every 1.7 microm from the stratum corneum surface to the first viable layer (stratum granulosum). Acidic microdomains (average pH 6.0) of variable size (~1 microm in diameter with variable length) are detected within the extracellular matrix of the stratum corneum, whereas the intracellular space of the corneocytes in mid-stratum corneum (25 microm diameter) approaches neutrality (average pH 7.0). The surface is acidic. The average pH of the stratum corneum increases with depth because of a decrease in the ratio of acidic to neutral regions within the stratum corneum. The data definitively show that the stratum corneum acid mantle results from the presence of aqueous acidic pockets within the lipid-rich extracellular matrix.     

CD44 interaction with Na+-H+ exchanger (NHE1) creates acidic microenvironments leading to hyaluronidase-2 and cathepsin B activation and breast tumor cell invasion

We have explored CD44 (a hyaluronan (HA) receptor) interaction with a Na(+)-H(+) exchanger (NHE1) and hyaluronidase-2 (Hyal-2) during HA-induced cellular signaling in human breast tumor cells (MDA-MB-231 cell line). Immunological analyses demonstrate that CD44s (standard form) and two signaling molecules (NHE1 and Hyal-2) are closely associated in a complex in MDA-MB-231 cells. These three proteins are also significantly enriched in cholesterol and ganglioside-containing lipid rafts, characterized as caveolin and flotillin-rich plasma membrane microdomains. The binding of HA to CD44 activates Na(+)-H(+) exchange activity which, in turn, promotes intracellular acidification and creates an acidic extracellular matrix environment. This leads to Hyal-2-mediated HA catabolism, HA modification, and cysteine proteinase (cathepsin B) activation resulting in breast tumor cell invasion. In addition, we have observed the following: (i) HA/CD44-activated Rho kinase (ROK) mediates NHE1 phosphorylation and activity, and (ii) inhibition of ROK or NHE1 activity (by treating cells with a ROK inhibitor, Y27632, or NHE1 blocker, S-(N-ethyl-N-isopropyl) amiloride, respectively) blocks NHE1 phosphorylation/Na(+)-H(+) exchange activity, reduces intracellular acidification, eliminates the acidic environment in the extracellular matrix, and suppresses breast tumor-specific behaviors (e.g. Hyal-2-mediated HA modification, cathepsin B activation, and tumor cell invasion). Finally, down-regulation of CD44 or Hyal-2 expression (by treating cells with CD44 or Hyal-2-specific small interfering RNAs) not only inhibits HA-mediated CD44 signaling (e.g. ROK-mediated Na(+)-H(+) exchanger reaction and cellular pH changes) but also impairs oncogenic events (e.g. Hyal-2 activity, hyaluronan modification, cathepsin B activation, and tumor cell invasion). Taken together, our results suggest that CD44 interaction with a ROK-activated NHE1 (a Na(+)-H(+) exchanger) in cholesterol/ganglioside-containing lipid rafts plays a pivotal role in promoting intracellular/extracellular acidification required for Hyal-2 and cysteine proteinase-mediated matrix degradation and breast cancer progression.     

pH microdomains in oligodendrocytes

Oligodendrocytes (OLs) are cells that produce myelin in the central nervous system. Here we use ratiometric pH indicator dye to analyze intracellular pH in OLs in culture. The results reveal alkaline microdomains, which predominate in the perikaryon and proximal dendrites, and acidic microdomains, which predominate in distal dendrites. Spatial nonuniformity of pH is generated by differential subcellular distribution of Na(+)/H(+) exchanger (NHE), which is localized in a punctate distribution in the perikaryon and proximal processes, Na(+)/HCO(3)(-) cotransporter (NBC), which is localized in a punctate distribution in distal dendrites, and carbonic anhydrase isotype II (CAII), which is colocalized with either NHE or NBC. Inhibition of NHE activity by amiloride inhibits regeneration of alkaline microdomains after cytoplasmic acidification, whereas the inhibition of CAII activity with ethoxyzolamide inhibits acidification of dendrites. Fluorescence correlation spectroscopy analysis of CAII microinjected into OLs reveals freely diffusing protein throughout the cell as well as protein associated predominantly with NHE in the perikaryon and predominantly with NBC in the dendrites. Alkaline and acidic microdomains could be generated by transport metabolons consisting of CAII associated with NHE or NBC, respectively. This study provides the first evidence for pH microdomains in cells and describes a mechanism for how they are generated.     

Na+/H+ Exchangers and RhoA Regulate Acidic Extracellular pH-Induced Lysosome Trafficking in Prostate Cancer Cells

Acidic extracellular pH (pHe) is a common feature of the tumor microenvironment and has been implicated in tumor invasion through the induction of protease secretion. Since lysosomes constitute the major storehouse of cellular proteases, the trafficking of lysosomes to the cell periphery may be required in order to secrete proteases. We demonstrate that a pHe of 6.4-6.8 induced the trafficking of lysosomes to membrane protrusions in the cell periphery. This trafficking event depended upon the PI3K pathway, the GTPase RhoA and sodium-proton exchange activity, resulting in lysosomal exocytosis. Acidic pHe induced a cytoplasmic acidification (although cytoplasmic acidification was not sufficient for acidic pHe-induced lysosome trafficking and exocytosis) and inhibition of NHE activity with the amiloride derivative, EIPA or the anti-diabetic agent troglitazone prevented lysosome trafficking to the cell periphery. Interestingly, using the more specific NHE1 and NHE3 inhibitors, cariporide and s3226 respectively, we show that multiple NHE isoforms are involved in acidic pHe-induced lysosome trafficking and exocytosis. Moreover, in cells expressing NHE1 shRNA, although basal NHE activity was decreased, lysosomes still underwent acidic pHe-induced trafficking, suggesting compensation by other NHE family members. Together these data implicate proton exchangers, especially NHE1 and NHE3, in acidic pHe-induced lysosome trafficking and exocytosis.     

pH nanoenvironment at the surface of single melanoma cells.

Extracellular pH and the Na(+)/H(+) exchanger (NHE1) modulate tumor cell migration. Yet, the pH nanoenvironment at the outer surface of the cell membrane (pH(em)) where cell/matrix interaction occurs and matrix metalloproteinases work was never measured. We present a method to measure this pH nanoenvironment using proton-sensitive dyes to label the outer leaflet of the plasma membrane or the glycocalyx of human melanoma cells. Polarized cells generate an extracellular proton gradient at their surface that increases from the rear end to the leading edge of the lamellipodium along the direction of movement. This gradient collapses upon NHE1 inhibition by HOE642. NHE1 stimulation by intracellular acidification increases the difference in pH(em) between the tips of lamellipodia and the cell body in a Na(+) dependent way. Thus, cells create a pH nanoenvironment that promotes cell migration by facilitating cell adhesion at their front and the release of cell/matrix contacts at their rear part.     

sPLA2 and the epidermal barrier

The mammalian epidermis provides both an interface and a protective barrier between the organism and its environment. Lipid, processed into water-impermeable bilayers between the outermost layers of the epidermal cells, forms the major barrier that prevents water from exiting the organism, and also prevents toxins and infectious agents from entering. The secretory phospholipase 2 (sPLA2) enzymes control important processes in skin and other organs, including inflammation and differentiation. sPLA2 activity contributes to epidermal barrier formation and homeostasis by generating free fatty acids, which are required both for formation of lamellar membranes and also for acidification of the stratum corneum (SC). sPLA2 is especially important in controlling SC acidification and establishment of an optimum epidermal barrier during the first postnatal week. Several sPLA2 isoforms are present in the epidermis. We find that two of these isoforms, sPLA2 IIA and sPLA2 IIF, localize to the upper stratum granulosum and increase in response to experimental barrier perturbation. sPLA2F^−/− mice also demonstrate a more neutral SC pH than do their normal littermates, and their initial recovery from barrier perturbation is delayed. These findings confirm that sPLA2 enzymes perform important roles in epidermal development, and suggest that the sPLA2IIF isoform may be central to SC acidification and barrier function. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

Differentiation-associated expression of ceramidase isoforms in cultured keratinocytes and epidermis

Ceramides (Cers) accumulate within the interstices of the outermost epidermal layers, or stratum corneum (SC), where they represent critical components of the epidermal permeability barrier. Although the SC contains substantial sphingol, indicative of ceramidase (CDase) activity, which CDase isoforms are expressed in epidermis remains unresolved. We hypothesized here that CDase isoforms are expressed within specific epidermal compartments in relation to functions that localize to these layers. Keratinocytes/epidermis express all five known CDase isoforms, of which acidic and alkaline CDase activities increase significantly with differentiation, persisting into the SC. Conversely, neutral and phytoalkaline CDase activities predominate in proliferating keratinocytes. These differentiation-associated changes in isoform activity/protein are attributed to corresponding, differentiation-associated changes in mRNA levels (by quantitative RT-PCR). Although four of the five known CDase isoforms are widely expressed in cutaneous and extracutaneous tissues, alkaline CDase-1 occurs almost exclusively in epidermis. These results demonstrate large, differentiation-associated, and tissue-specific variations in the expression and activities of all five CDase isoforms. Because alkaline CDase-1 and acidic CDase are selectively upregulated in the differentiated epidermal compartment, they could regulate functions that localize to the distal epidermis, such as permeability barrier homeostasis and antimicrobial defense.     

Intracellular acidification is associated with changes in free cytosolic calcium and inhibition of action potentials in rat trigeminal ganglion

The effect of intracellular acidification and subsequent pH recovery in sensory neurons has not been well characterized. We have studied the mechanisms underlying Ca(2+)-induced acidification and subsequent recovery of intracellular pH (pH(i)) in rat trigeminal ganglion neurons and report their effects on neuronal excitability. Glutamate (500 μM) and capsaicin (1 μM) increased intracellular Ca(2+) concentration ([Ca(2+)](i)) with a following decrease in pH(i). The recovery of [Ca(2+)](i) to the prestimulus level was inhibited by LaCl(3) (1 mM) and o-vanadate (10 mM), a plasma membrane Ca(2+)/ATPase (PMCA) inhibitor. Removal of extracellular Ca(2+) also completely inhibited the acidification induced by capsaicin. TRPV1 was expressed only in small and medium sized trigeminal ganglion neurons. mRNAs for Na(+)/H(+) exchanger type 1 (NHE1), pancreatic Na(+)-HCO(3)(-) cotransporter type 1 (pNBC1), NBC3, NBC4, and PMCA types 1-3 were detected by RT-PCR. pH(i) recovery was significantly inhibited by pretreatment with NHE1 or pNBC1 siRNA. We found that the frequency of action potentials (APs) was dependent on pH(i). Application of the NHE1 inhibitor 5'-(N-ethyl-N-isopropyl) amiloride (5 μM) or the pNBC1 inhibitor 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid (500 μM) delayed pH(i) recovery and decreased AP frequency. Simultaneous application of 5'-(N-ethyl-N-isopropyl) amiloride and 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid almost completely inhibited APs. In summary, our results demonstrate that the rise in [Ca(2+)](i) in sensory neurons by glutamate and capsaicin causes intracellular acidification by activation of PMCA type 3, that the pH(i) recovery from acidification is mediated by membrane transporters NHE1 and pNBC1 specifically, and that the activity of these transporters has direct consequences for neuronal excitability.     

Dimerization is crucial for the function of the Na+/H+ exchanger NHE1

The Na(+)/H(+) exchanger 1 (NHE1) exists as a homo-dimer in the plasma membranes. In the present study, we have investigated the functional significance of the dimerization, using two nonfunctional NHE1 mutants, surface-expression-deficient G309V and transport-deficient E262I. Biochemical and immunocytochemical experiments revealed that these NHE1 mutants are capable of interacting with the wild-type NHE1 and, thus, forming a heterodimer. Expression of G309V retained the wild-type NHE1 to the ER membranes, suggesting that NHE1 would first form a dimer in the ER. On the other hand, expression of E262I markedly reduced the exchange activity of the wild-type NHE1 through an acidic shift in the intracellular pH (pH(i)) dependence, suggesting that dimerization is required for exchange activity in the physiological pH(i) range. However, a dominant-negative effect of E262I was not detected when exchange activity was measured at acidic pH(i), implying that one active subunit is sufficient to catalyze ion transport when the intracellular H(+) concentration is sufficiently high. Furthermore, intermolecular cysteine cross-linking at extracellular position Ser(375) with a bifunctional sulfhydryl reagent dramatically inhibited exchange activity mainly by inducing the acidic shift of pH(i) dependence and abolished extracellular stimuli-induced activation of NHE1 without causing a large change in the affinities for extracellular Na(+) or an inhibitor EIPA. Because monofunctional sulfhydryl regents had no effect, it is likely that cross-linking inhibited the activity of NHE1 by restricting a coupled motion between the two subunits during transport. Taken together, these data support the view that dimerization of two active subunits are required for NHE1 to possess the exchange activity in the neutral pH(i) range, although each subunit is capable of catalyzing transport in the acidic pH(i) range.     

Extracellular acidification induces human neutrophil activation

In the current work, we evaluated the effect of extracellular acidification on neutrophil physiology. Neutrophils suspended in bicarbonate-buffered RPMI 1640 medium adjusted to acidic pH values (pH 6.5-7.0) underwent: 1) a rapid transient increase in intracellular free calcium concentration levels; 2) an increase in the forward light scattering properties; and 3) the up-regulation of surface expression of CD18. By contrast, extracellular acidosis was unable to induce neither the production of H2O2 nor the release of myeloperoxidase. Acidic extracellular pH also modulated the functional profile of neutrophils in response to conventional agonists such as FMLP, precipiting immune complexes, and opsonized zymosan. It was found that not only calcium mobilization, shape change response, and up-regulation of CD18 expression but also production of H2O2 and release of myeloperoxidase were markedly enhanced in neutrophils stimulated in acidic pH medium. Moreover, extracellular acidosis significantly delayed neutrophil apoptosis and concomitantly extended neutrophil functional lifespan. Extracellular acidification induced an immediate and abrupt fall in the intracellular pH, which persisted over the 240-s analyzed. A similar abrupt drop in the intracellular pH was detected in cells suspended in bicarbonate-supplemented PBS but not in those suspended in bicarbonate-free PBS. A role for intracellular acidification in neutrophil activation is suggested by the fact that only neutrophils suspended in bicarbonate-buffered media (i.e., RPMI 1640 and bicarbonate-supplemented PBS) underwent significant shape changes in response to extracellular acidification. Together, our results support the notion that extracellular acidosis may intensify acute inflammatory responses by inducing neutrophil activation as well as by delaying spontaneous apoptosis and extending neutrophil functional lifespan.     

Activation of AMP-activated Protein Kinase Regulates Hippocampal Neuronal pH by Recruiting Na+/H+ Exchanger NHE5 to the Cell Surface

Strict regulation of intra- and extracellular pH is an important determinant of nervous system function as many voltage-, ligand-, and H⁺-gated cationic channels are exquisitely sensitive to transient fluctuations in pH elicited by neural activity and pathophysiologic events such as hypoxia-ischemia and seizures. Multiple Na⁺/H⁺ exchangers (NHEs) are implicated in maintenance of neural pH homeostasis. However, aside from the ubiquitous NHE1 isoform, their relative contributions are poorly understood. NHE5 is of particular interest as it is preferentially expressed in brain relative to other tissues. In hippocampal neurons, NHE5 regulates steady-state cytoplasmic pH, but intriguingly the bulk of the transporter is stored in intracellular vesicles. Here, we show that NHE5 is a direct target for phosphorylation by the AMP-activated protein kinase (AMPK), a key sensor and regulator of cellular energy homeostasis in response to metabolic stresses. In NHE5-transfected non-neuronal cells, activation of AMPK by the AMP mimetic AICAR or by antimycin A, which blocks aerobic respiration and causes acidification, increased cell surface accumulation and activity of NHE5, and elevated intracellular pH. These effects were effectively blocked by the AMPK antagonist compound C, the NHE inhibitor HOE694, and mutation of a predicted AMPK recognition motif in the NHE5 C terminus. This regulatory pathway was also functional in primary hippocampal neurons, where AMPK activation of NHE5 protected the cells from sustained antimycin A-induced acidification. These data reveal a unique role for AMPK and NHE5 in regulating the pH homeostasis of hippocampal neurons during metabolic stress.     

Beta-glucocerebrosidase activity in mammalian stratum corneum

Although previous studies have demonstrated a crucial role for the enzyme beta-glucocerebrosidase (GlcCer'ase) in the final steps of membrane structural maturation in mammalian stratum cornuem (SC) and epidermal homeostasis, the precise in vivo localization of GlcCer'ase activity and protein is not known. Here, we developed a fluorogenic in situ assay on histologic sections (zymography) to elucidate the in vivo distribution of GlcCer'ase activity, and further characterized and localized the SC GlcCer'ase activity in vitro. The zymographic technique revealed higher GlcCer'ase activity in upper stratum granulosum and SC, both in murine and human SC; activity that was both inhibited by conduritol B epoxide, a specific GlcCer'ase inhibitor, and pH-dependent; i.e., present at pH 5.2, and absent or significantly reduced at neutral pH (7.4), consistent with the known pH optimum for epidermal GlcCer'ase in vitro. Immunohistochemical staining for GlcCer'ase protein showed enhanced fluorescent signal in the outer layers of human epidermis, concentrated at the apex and margins of stratum granulosum and lower SC. Moreover, in extracts from individual epidermal layers, GlcCer'ase activity was present throughout murine epidermis, with the highest activity in the SC, peaking in the lower-to-mid-SC. The SC activity was stimulated >10-fold by sodium taurocholate, and inhibited by bromoconduritol B epoxide. Finally, isolated membrane couplets, prepared from SC sheets, also demonstrated significant GlcCer'ase activity. These data localize GlcCer'ase activity to the outer epidermis by three different techniques, and support the role of this enzyme in extracellular processing of glucosylceramides to ceramides, required for permeability barrier maturation and function.     

Towards a Quantitative Theory of Epidermal Calcium Profile Formation in Unwounded Skin

We propose and mathematically examine a theory of calcium profile formation in unwounded mammalian epidermis based on: changes in keratinocyte proliferation, fluid and calcium exchange with the extracellular fluid during these cells’ passage through the epidermal sublayers, and the barrier functions of both the stratum corneum and tight junctions localised in the stratum granulosum. Using this theory, we develop a mathematical model that predicts epidermal sublayer transit times, partitioning of the epidermal calcium gradient between intracellular and extracellular domains, and the permeability of the tight junction barrier to calcium ions. Comparison of our model’s predictions of epidermal transit times with experimental data indicates that keratinocytes lose at least 87% of their volume during their disintegration to become corneocytes. Intracellular calcium is suggested as the main contributor to the epidermal calcium gradient, with its distribution actively regulated by a phenotypic switch in calcium exchange between keratinocytes and extracellular fluid present at the boundary between the stratum spinosum and the stratum granulosum. Formation of the extracellular calcium distribution, which rises in concentration through the stratum granulosum towards the skin surface, is attributed to a tight junction barrier in this sublayer possessing permeability to calcium ions that is less than 15 nm s⁻¹ in human epidermis and less than 37 nm s⁻¹ in murine epidermis. Future experimental work may refine the presented theory and reduce the mathematical uncertainty present in the model predictions.     

Regulation of dendritic spine growth through activity-dependent recruitment of the brain-enriched Na⁺/H⁺ exchanger NHE5

Subtle changes in cellular and extracellular pH within the physiological range have profound impacts on synaptic activities. However, the molecular mechanisms underlying local pH regulation at synapses and their influence on synaptic structures have not been elucidated. Dendritic spines undergo dynamic structural changes in response to neuronal activation, which contributes to induction and long-term maintenance of synaptic plasticity. Although previous studies have indicated the importance of cytoskeletal rearrangement, vesicular trafficking, cell signaling, and adhesion in this process, much less is known about the involvement of ion transporters. In this study we demonstrate that N-methyl-D-aspartate (NMDA) receptor activation causes recruitment of the brain-enriched Na(+)/H(+) exchanger NHE5 from endosomes to the plasma membrane. Concomitantly, real-time imaging of green fluorescent protein-tagged NHE5 revealed that NMDA receptor activation triggers redistribution of NHE5 to the spine head. We further show that neuronal activation causes alkalinization of dendritic spines following the initial acidification, and suppression of NHE5 significantly retards the activity-induced alkalinization. Perturbation of NHE5 function induces spontaneous spine growth, which is reversed by inhibition of NMDA receptors. In contrast, overexpression of NHE5 inhibits spine growth in response to neuronal activity. We propose that NHE5 constrains activity-dependent dendritic spine growth via a novel, pH-based negative-feedback mechanism.     

Increased tolerance to oxygen and glucose deprivation in astrocytes from Na(+)/H(+) exchanger isoform 1 null mice

The ubiquitously expressed Na(+)/H(+) exchanger isoform 1 (NHE1) functions as a major intracellular pH (pH(i)) regulatory mechanism in many cell types, and in some tissues its activity may contribute to ischemic injury. In the present study, cortical astrocyte cultures from wild-type (NHE1(+/+)) and NHE1-deficient (NHE1(-/-)) mice were used to investigate the role of NHE1 in pH(i) recovery and ischemic injury in astrocytes. In the absence of HCO(3)(-), the mean resting pH(i) levels were 6.86 +/- 0.03 in NHE1(+/+) astrocytes and 6.53 +/- 0.04 in NHE1(-/-) astrocytes. Removal of extracellular Na(+) or blocking of NHE1 activity by the potent NHE1 inhibitor HOE-642 significantly reduced the resting level of pH(i) in NHE1(+/+) astrocytes. NHE1(+/+) astrocytes exhibited a rapid pH(i) recovery (0.33 +/- 0.08 pH unit/min) after NH(4)Cl prepulse acid load. The pH(i) recovery in NHE1(+/+) astrocytes was reversibly inhibited by HOE-642 or removal of extracellular Na(+). In NHE1(-/-) astrocytes, the pH(i) recovery after acidification was impaired and not affected by either Na(+)-free conditions or HOE-642. Furthermore, 2 h of oxygen and glucose deprivation (OGD) led to an approximately 80% increase in pH(i) recovery rate in NHE1(+/+) astrocytes. OGD induced a 5-fold rise in intracellular [Na(+)] and 26% swelling in NHE1(+/+) astrocytes. HOE-642 or genetic ablation of NHE1 significantly reduced the Na(+) rise and swelling after OGD. These results suggest that NHE1 is the major pH(i) regulatory mechanism in cortical astrocytes and that ablation of NHE1 in astrocytes attenuates ischemia-induced disruption of ionic regulation and swelling.     

Mechanism of acid adaptation of a fish living in a pH 3.5 lake

Despite unfavorable conditions, a single species of fish, Osorezan dace, lives in an extremely acidic lake (pH 3.5) in Osorezan, Aomori, Japan. Physiological studies have established that this fish is able to prevent acidification of its plasma and loss of Na(+). Here we show that these abilities are mainly attributable to the chloride cells of the gill, which are arranged in a follicular structure and contain high concentrations of Na(+)-K(+)-ATPase, carbonic anhydrase II, type 3 Na(+)/H(+) exchanger (NHE3), type 1 Na(+)-HCO(3)(-) cotransporter, and aquaporin-3, all of which are upregulated on acidification. Immunohistochemistry established their chloride cell localization, with NHE3 at the apical surface and the others localized to the basolateral membrane. These results suggest a mechanism by which Osorezan dace adapts to its acidic environment. Most likely, NHE3 on the apical side excretes H(+) in exchange for Na(+), whereas the electrogenic type 1 Na(+)-HCO(3)(-) cotransporter in the basolateral membrane provides HCO(3)(-) for neutralization of plasma using the driving force generated by Na(+)-K(+)-ATPase and carbonic anhydrase II. Increased expression of glutamate dehydrogenase was also observed in various tissues of acid-adapted dace, suggesting a significant role of ammonia and bicarbonate generated by glutamine catabolism.     

Oxidative stress decreases pHi and Na(+)/H(+) exchange and increases excitability of solitary complex neurons from rat brain slices

Putative chemoreceptors in the solitary complex (SC) are sensitive to hypercapnia and oxidative stress. We tested the hypothesis that oxidative stress stimulates SC neurons by a mechanism independent of intracellular pH (pH_i). pH_i was measured by using ratiometric fluorescence imaging microscopy, utilizing either the pH-sensitive fluorescent dye BCECF or, during whole cell recordings, pyranine in SC neurons in brain stem slices from rat pups. Oxidative stress decreased pH_i in 270 of 436 (62%) SC neurons tested. Chloramine-T (CT), N-chlorosuccinimide (NCS), dihydroxyfumaric acid, and H₂O₂ decreased pH_i by 0.19 ± 0.007, 0.20 ± 0.015, 0.15 ± 0.013, and 0.08 ± 0.002 pH unit, respectively. Hypercapnia decreased pH_i by 0.26 ± 0.006 pH unit (n = 95). The combination of hypercapnia and CT or NCS had an additive effect on pH_i, causing a 0.42 ± 0.03 (n = 21) pH unit acidification. CT slowed pH_i recovery mediated by Na⁺/H⁺ exchange (NHE) from NH₄Cl-induced acidification by 53% (n = 20) in -buffered medium and by 58% (n = 10) in HEPES-buffered medium. CT increased firing rate in 14 of 16 SC neurons, and there was no difference in the firing rate response to CT with or without a corresponding change in pH_i. These results indicate that oxidative stress 1) decreases pH_i in some SC neurons, 2) together with hypercapnia has an additive effect on pH_i, 3) partially inhibits NHE, and 4) directly affects excitability of CO₂/H⁺-chemosensitive SC neurons independently of pH_i changes. These findings suggest that oxidative stress acidifies SC neurons in part by inhibiting NHE, and this acidification may contribute ultimately to respiratory control dysfunction. hyperoxic hyperventilation; O₂ toxicity; pH regulation; brain stem; reactive oxygen species     

The Na+/H+‐exchanger (NHE1) generates pH nanodomains at focal adhesions

Many tumor cells are characterized by an increased net acid production. They extrude the excess protons mainly through the Na⁺/H⁺‐exchanger NHE1. An increased NHE1 activity elevates the metastatic potential of tumor cells. Cell migration, a key step in the metastatic cascade, requires the formation and release of integrin‐mediated cell–matrix contacts (focal adhesions). As NHE1 has been localized to focal adhesion sites, the present study tests the hypothesis that NHE1 generates measurable pH nanodomains right at focal adhesions. In order to ratiometrically measure pH close to the plasma membrane, we established a novel application of the total internal reflection fluorescence microscopy (TIRFM). Human melanoma cells were transfected with DsRed2‐paxillin to identify focal adhesion sites. The pH‐sensitive dyes BCECF and WGA‐fluorescein were used to measure the submembranous cytosolic and the pericellular pH, respectively. Distinct pH nanodomains were found at focal adhesions, particularly at those located at the cell front, where NHE1 was concentrated. These sites featured a remarkably alkaline cytosolic and an acidic pericellular pH and thus a much steeper proton gradient across the plasma membrane compared to the rest of the cell. The generation of pH nanodomains could be assigned to NHE1‐mediated H⁺ export because such pH domains could not be detected in NHE1‐deficient cells. Given that both integrin avidity and mechanisms contributing to adhesion turnover are pH‐sensitive, we propose that pH nanodomains at focal adhesions, locally created and maintained by NHE1 activity especially at the cell front, modulate adhesion dynamics in migrating cells. J. Cell. Physiol. 228: 1351–1358, 2013. © 2012 Wiley Periodicals, Inc.     

Protons extruded by NHE1: digestive or glue?

Many physiological and pathophysiological processes, such as embryogenesis, immune defense, wound healing, or metastasis, are based on cell migration and invasion. The activity of the ubiquitously expressed NHE1 isoform of the plasma membrane Na(+)/H(+) exchanger is one of the requirements for directed locomotion of migrating cells. The mechanisms by which NHE1 is involved in cell migration are multiple. NHE1 contributes to cell migration by affecting the cell volume, by regulating the intracellular pH and thereby the assembly and activity of cytoskeletal elements, by anchoring the cytoskeleton to the plasma membrane, by the organization of signal transduction and by regulating gene expression. The present review focuses on two additional, extracellular mechanisms by which NHE1 activity contributes to cell migration and invasion. Protons extruded by the NHE1 lead to local, extracellular acidification which, on the one hand, can create pH optima needed for the activity of proteinases at invadopodia/podosomes necessary for extracellular matrix digestion and, on the other hand, facilitates cell/matrix interaction and adhesion at the cell front.