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The expansion of cancer cell mass in solid tumors generates a harsh environment characterized by dynamically varying levels of acidosis, hypoxia, and nutrient deprivation. Because acidosis inhibits glycolytic metabolism and hypoxia inhibits oxidative phosphorylation, cancer cells that survive and grow in these environments must rewire their metabolism and develop a high degree of metabolic plasticity to meet their energetic and biosynthetic demands. Cancer cells frequently upregulate pathways enabling the uptake and utilization of lipids and other nutrients derived from dead or recruited stromal cells, and in particular lipid uptake is strongly enhanced in acidic microenvironments. The resulting lipid accumulation and increased reliance on β-oxidation and mitochondrial metabolism increase susceptibility to oxidative stress, lipotoxicity, and ferroptosis, in turn driving changes that may mitigate such risks. The spatially and temporally heterogeneous tumor microenvironment thus selects for invasive, metabolically flexible, and resilient cancer cells capable of exploiting their local conditions and of seeking out more favorable surroundings. This phenotype relies on the interplay between metabolism, acidosis, and oncogenic mutations, driving metabolic signaling pathways such as peroxisome proliferator-activated receptors (PPARs). Understanding the particular vulnerabilities of such cells may uncover novel therapeutic liabilities of the most aggressive cancer cells.

Na + /H + exchangers play pivotal roles in the control of cell and tissue pH by mediating the electroneutral exchange of Na + and H + across cellular membranes. They belong to an ancient family of highly evolutionarily conserved proteins, and they play essential physiological roles in all phyla. In this review, we focus on the mammalian Na + /H + exchangers (NHEs), the solute carrier (SLC) 9 family. This family of electroneutral transporters constitutes three branches: SLC9A, -B, and -C. Within these, each isoform exhibits distinct tissue expression profiles, regulation, and physiological roles. Some of these transporters are highly studied, with hundreds of original articles, and some are still only rudimentarily understood. In this review, we present and discuss the pioneering original work as well as the current state-of-the-art research on mammalian NHEs. We aim to provide the reader with a comprehensive view of core knowledge and recent insights into each family member, from gene organization over protein structure and regulation to physiological and pathophysiological roles. Particular attention is given to the integrated physiology of NHEs in the main organ systems. We provide several novel analyses and useful overviews, and we pinpoint main remaining enigmas, which we hope will inspire novel research on these highly versatile proteins.

The mechanisms linking cell volume sensing to volume regulation in mammalian cells remain incompletely understood. Here, we test the hypothesis that activation of nonreceptor tyrosine kinases Src, focal adhesion kinase (FAK), and Janus kinase-2 (Jak2) occurs after osmotic shrinkage of NIH3T3 fibroblasts and contributes to volume regulation by activation of NKCC1. FAK phosphorylation at Tyr397, Tyr576/577, and Tyr861 was increased rapidly after exposure to hypertonic (575 mOsm) saline, peaking after 10 (Tyr397, Tyr576/577) and 10-30 min (Tyr861). Shrinkage-induced Src family kinase autophosphorylation (pTyr416-Src) was induced after 2-10 min, and immunoprecipitation indicated that this reflected phosphorylation of Src itself, rather than Fyn and Yes. Phosphorylated Src and FAK partly colocalized with vinculin, a focal adhesion marker, after hypertonic shrinkage. The Src inhibitor pyrazolopyrimidine-2 (PP2, 10 μM) essentially abolished shrinkage-induced FAK phosphorylation at Tyr576/577 and Tyr861, yet not at Tyr397, and inhibited shrinkage-induced NKCC1 activity by ∼50%. The FAK inhibitor PF-573,228 augmented shrinkage-induced Src phosphorylation, and inhibited shrinkage-induced NKCC1 activity by ∼15%. The apparent role of Src in NKCC1 activation did not reflect phosphorylation of myosin light chain kinase (MLC), which was unaffected by shrinkage and by PP2, but may involve Jak2, a known target of Src, which was rapidly activated by osmotic shrinkage and inhibited by PP2. Collectively, our findings suggest a major role for Src and possibly the Jak2 axis in shrinkage-activation of NKCC1 in NIH3T3 cells, whereas no evidence was found for major roles for FAK and MLC in this process., (Copyright © 2015 the American Physiological Society.)

Hyperosmotic stress initiates several adaptive responses, including the remodeling of the cytoskeleton. Besides maintaining structural integrity, the cytoskeleton has emerged as an important regulator of gene transcription. Myocardin-related transcription factor (MRTF), an actin-regulated coactivator of serum response factor, is a major link between the actin skeleton and transcriptional control. We therefore investigated whether MRTF is regulated by hyperosmotic stress. Here we show that hypertonicity induces robust, rapid, and transient translocation of MRTF from the cytosol to the nucleus in kidney tubular cells. We found that the hyperosmolarity-triggered MRTF translocation is mediated by the RhoA/Rho kinase (ROK) pathway. Moreover, the Rho guanine nucleotide exchange factor GEF-H1 is activated by hyperosmotic stress, and it is a key contributor to the ensuing RhoA activation and MRTF translocation, since siRNA-mediated GEF-H1 downregulation suppresses these responses. While the osmotically induced RhoA activation promotes nuclear MRTF accumulation, the concomitant activation of p38 MAP kinase mitigates this effect. Moderate hyperosmotic stress (600 mosM) drives MRTF-dependent transcription through the cis-element CArG box. Silencing or pharmacological inhibition of MRTF prevents the osmotic stimulation of CArG-dependent transcription and renders the cells susceptible to osmotic shock-induced structural damage. Interestingly, strong hyperosmolarity promotes proteasomal degradation of MRTF, concomitant with apoptosis. Thus, MRTF is an osmosensitive and osmoprotective transcription factor, whose intracellular distribution is regulated by the GEF-H1/RhoA/ROK and p38 pathways. However, strong osmotic stress destabilizes MRTF, concomitant with apoptosis, implying that hyperosmotically induced cell death takes precedence over epithelial-myofibroblast transition, a potential consequence of MRTF-mediated phenotypic reprogramming.

Ca(+) signaling plays a crucial role in control of cell cycle progression, but the understanding of the dynamics of Ca(2+) influx and release of Ca(2+) from intracellular stores during the cell cycle is far from complete. The aim of the present study was to investigate the role of the free extracellular Ca(2+) concentration ([Ca(2+)](o)) in cell proliferation, the pattern of changes in the free intracellular Ca(2+) concentration ([Ca(2+)](i)) during cell cycle progression, and the role of the transient receptor potential (TRP)C1 in these changes as well as in cell cycle progression and cell volume regulation. In Ehrlich Lettré Ascites (ELA) cells, [Ca(2+)](i) decreased significantly, and the thapsigargin-releasable Ca(2+) pool in the intracellular stores increased in G(1) as compared with G(0). Store-depletion-operated Ca(2+) entry (SOCE) and TRPC1 protein expression level were both higher in G(1) than in G(0) and S phase, in parallel with a more effective volume regulation after swelling [regulatory volume decrease (RVD)] in G(1) as compared with S phase. Furthermore, reduction of [Ca(2+)](o), as well as two unspecific SOCE inhibitors, 2-APB (2-aminoethyldiphenyl borinate) and SKF96365 (1-(β-[3-(4-methoxy-phenyl)propoxyl-4-methoxyphenethyl)1H-imidazole-hydrochloride), inhibited ELA cell proliferation. Finally, Madin-Darby canine kidney cells in which TRPC1 was stably silenced [TRPC1 knockdown (TRPC1-KD) MDCK] exhibited reduced SOCE, slower RVD, and reduced cell proliferation compared with mock controls. In conclusion, in ELA cells, SOCE and TRPC1 both seem to be upregulated in G(1) as compared with S phase, concomitant with an increased rate of RVD. Furthermore, TRPC1-KD MDCK cells exhibit decreased SOCE, decreased RVD, and decreased proliferation, suggesting that, at least in certain cell types, TRPC1 is regulated during cell cycle progression and is involved in SOCE, RVD, and cell proliferation.

Cell polarization along the axis of movement is required for migration. The localization of proteins and regulators of the migratory machinery to either the cell front or its rear results in a spatial asymmetry enabling cells to simultaneously coordinate cell protrusion and retraction. Protons might function as such unevenly distributed regulators as they modulate the interaction of focal adhesion proteins and components of the cytoskeleton in vitro. However, an intracellular pH (pH(i)) gradient reflecting a spatial asymmetry of protons has not been shown so far. One major regulator of pH(i), the Na(+)/H(+) exchanger NHE1, is essential for cell migration and accumulates at the cell front. Here, we test the hypothesis that the uneven distribution of NHE1 activity creates a pH(i) gradient in migrating cells. Using the pH-sensitive fluorescent dye BCECF, pH(i) was measured in five cell lines (MV3, B16V, NIH3T3, MDCK-F1, EA.hy926) along the axis of movement. Differences in pH(i) between the front and the rear end (ΔpH(i) front-rear) were present in all cell lines, and inhibition of NHE1 either with HOE642 or by absence of extracellular Na(+) caused the pH(i) gradient to flatten or disappear. In conclusion, pH(i) gradients established by NHE1 activity exist along the axis of movement.

Channels and transporters of monovalent ions are increasingly suggested as putative anticarcinogenic targets. However, the mechanisms involved in modulation of proliferation by monovalent ions are poorly understood. Here, we investigated the role of K+, Na+, and Cl(-) ions for the proliferation of Ehrlich Lettre ascites (ELA) cells. We measured the intracellular concentration of each ion in G(0), G(1), and S phases of the cell cycle following synchronization by serum starvation and release. We show that intracellular concentrations and content of Na+ and Cl(-) were reduced in the G(0)-G(1) phase transition, followed by an increased content of both ions in S phase concomitant with water uptake. The effect of substituting extracellular monovalent ions was investigated by bromodeoxyuridine incorporation and showed marked reduction after Na+ and Cl(-) substitution. In spectrofluorometric measurements with the pH-sensitive dye BCECF, substitution of Na+ was observed to upregulate the activity of the Na+/H+ exchanger NHE1 as well as of Na+-independent acid extrusion mechanisms, facilitating intracellular pH (pH(i)) recovery after acid loading and increasing pH(i). Results using the potential sensitive dye DiBaC4(3) showed a reduced Cl(-) conductance in S compared with G(1) followed by transmembrane potential (E(m)) hyperpolarization in S. Cl(-) substitution by impermeable anions strongly inhibited proliferation and increased free, intracellular Ca2+ ([Ca2+]i), whereas a more permeable anion had little effect. Western blots showed reduced chloride intracellular channel CLIC1 and chloride channel ClC-2 expression in the plasma membrane in S compared with G(1). Our results suggest that Na+ regulates ELA cell proliferation by regulating intracellular pH while Cl(-) may regulate proliferation by fine-tuning of E(m) in S phase and altered Ca2+ signaling.

The Ca(2+)-independent phospholipase A(2) VI (iPLA(2)-VI) and the Na(+)/H(+) exchanger isoform 1 (NHE1) are highly pH-sensitive proteins that exert both protective and detrimental effects in cardiac ischemia-reperfusion. Here, we investigated the role of extracellular pH (pH(o)) in ischemia-reperfusion injury and death and in regulation and function of iPLA(2)-VI and NHE1 under these conditions. HL-1 cardiomyocytes were exposed to simulated ischemia (SI; 0.5% O(2), 8 mM K(+), and 20 mM lactate) at pH(o) 6.0 and 7.4, with or without 4 or 8 h of reperfusion (SI/R). Cytochrome c release and caspase-3 activation were reduced after acidic compared with neutral SI, whereas necrotic death, estimated as glucose-6-phosphate dehydrogenase release, was similar in the two conditions. Inhibition of iPLA(2)-VI activity by bromoenol lactone (BEL) elicited cardiomyocyte necrosis during normoxia and after acidic, yet not after neutral, SI. The isoform-selective enantiomers R- and S-BEL both mimicked the effect of racemic BEL after acidic SI. In contrast, inhibition of NHE activity by EIPA had no significant effect on necrosis after SI. Both neutral and acidic SI were associated with a reversible loss of F-actin and cortactin integrity. Inhibition of iPLA(2)-VI disrupted F-actin, cortactin, and mitochondrial integrity, whereas inhibition of NHE slightly reduced stress fiber content. iPLA(2)-VIA and NHE1 mRNA levels were reduced during SI and upregulated in a pH(o)-dependent manner during SI/R. This also affected the subcellular localization of iPLA(2)-VIA. Thus, the mode of cell death and the roles and regulation of iPLA(2)-VI and NHE1 are at least in part determined by the pH(o) during SI. In addition to having clinically relevant implications, these findings can in part explain the contradictory results obtained from previous studies of iPLA(2)-VIA and NHE1 during cardiac I/R.

Hyperosmotic stress induces cytoskeleton reorganization and a net increase in cellular F-actin, but the underlying mechanisms are incompletely understood. Whereas de novo F-actin polymerization likely contributes to the actin response, the role of F-actin severing is unknown. To address this problem, we investigated whether hyperosmolarity regulates cofilin, a key actin-severing protein, the activity of which is inhibited by phosphorylation. Since the small GTPases Rho and Rac are sensitive to cell volume changes and can regulate cofilin phosphorylation, we also asked whether they might link osmostress to cofilin. Here we show that hyperosmolarity induced rapid, sustained, and reversible phosphorylation of cofilin in kidney tubular (LLC-PK1 and Madin-Darby canine kidney) cells. Hyperosmolarity-provoked cofilin phosphorylation was mediated by the Rho/Rho kinase (ROCK)/LIM kinase (LIMK) but not the Rac/PAK/LIMK pathway, because 1) dominant negative (DN) Rho and DN-ROCK but not DN-Rac and DN-PAK inhibited cofilin phosphorylation; 2) constitutively active (CA) Rho and CA-ROCK but not CA-Rac and CA-PAK induced cofilin phosphorylation; 3) hyperosmolarity induced LIMK-2 phosphorylation, and 4) inhibition of ROCK by Y-27632 suppressed the hypertonicity-triggered LIMK-2 and cofilin phosphorylation.We thenexamined whether cofilin and its phosphorylation play a role in the hypertonicity-triggered F-actin changes. Downregulation of cofilin by small interfering RNA increased the resting F-actin level and eliminated any further rise upon hypertonic treatment. Inhibition of cofilin phosphorylation by Y-27632 prevented the hyperosmolarity-provoked F-actin increase. Taken together, cofilin is necessary for maintaining the osmotic responsiveness of the cytoskeleton in tubular cells, and the Rho/ROCK/LIMK-mediated cofilin phosphorylation is a key mechanism in the hyperosmotic stress-induced F-actin increase.

The ability to control cell volume is pivotal for cell function. Cell volume perturbation elicits a wide array of signaling events, leading to protective (e.g., cytoskeletal rearrangement) and adaptive (e.g., altered expression of osmolyte transporters and heat shock proteins) measures and, in most cases, activation of volume regulatory osmolyte transport. After acute swelling, cell volume is regulated by the process of regulatory volume decrease (RVD), which involves the activation of KCl cotransport and of channels mediating K(+), Cl(-), and taurine efflux. Conversely, after acute shrinkage, cell volume is regulated by the process of regulatory volume increase (RVI), which is mediated primarily by Na(+)/H(+) exchange, Na(+)-K(+)-2Cl(-) cotransport, and Na(+) channels. Here, we review in detail the current knowledge regarding the molecular identity of these transport pathways and their regulation by, e.g., membrane deformation, ionic strength, Ca(2+), protein kinases and phosphatases, cytoskeletal elements, GTP binding proteins, lipid mediators, and reactive oxygen species, upon changes in cell volume. We also discuss the nature of the upstream elements in volume sensing in vertebrate organisms. Importantly, cell volume impacts on a wide array of physiological processes, including transepithelial transport; cell migration, proliferation, and death; and changes in cell volume function as specific signals regulating these processes. A discussion of this issue concludes the review.

Hyperosmotic shrinkage induces multiple cellular responses, including activation of volume-regulatory ion transport, cytoskeletal reorganization, and cell death. Here we investigated the possible roles of ezrin/radixin/moesin (ERM) proteins in these events. Osmotic shrinkage of Ehrlich Lettre ascites cells elicited the formation of long microvillus-like protrusions, rapid translocation of endogenous ERM proteins and green fluorescent protein-tagged ezrin to the cortical region including these protrusions, and Thr(567/564/558) (ezrin/radixin/moesin) phosphorylation of cortical ERM proteins. Reduced cell volume appeared to be the critical parameter in hypertonicity-induced ERM protein activation, whereas alterations in extracellular ionic strength or intracellular pH were not involved. A shrinkage-induced increase in the level of membrane-associated phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] appeared to play an important role in ERM protein activation, which was prevented after PtdIns(4,5)P(2) depletion by expression of the synaptojanin-2 phosphatase domain. While expression of constitutively active RhoA increased basal ERM phosphorylation, the Rho-Rho kinase pathway did not appear to be involved in shrinkage-induced ERM protein phosphorylation, which was also unaffected by the inhibition or absence of Na(+)/H(+) exchanger isoform (NHE1). Ezrin knockdown by small interfering RNA increased shrinkage-induced NHE1 activity, reduced basal and shrinkage-induced Rho activity, and attenuated the shrinkage-induced formation of microvillus-like protrusions. Hyperosmolarity-induced cell death was unaltered by ezrin knockdown or after phosphatidylinositol 3-kinase (PI3K) inhibition. In conclusion, ERM proteins are activated by osmotic shrinkage in a PtdIns(4,5)P(2)-dependent, NHE1-independent manner. This in turn mitigates the shrinkage-induced activation of NHE1, augments Rho activity, and may also contribute to F-actin rearrangement. In contrast, no evidence was found for the involvement of an NHE1-ezrin-PI3K-PKB pathway in counteracting shrinkage-induced cell death.

The involvement of group VI Ca(2+)-independent PLA(2)s (iPLA(2)-VI) in in vitro ischemia [oxygen and glucose deprivation (OGD)] in mouse C2C12 myotubes was investigated. OGD induced a time-dependent (0-6 h) increase in bromoenol lactone (BEL)-sensitive iPLA(2) activity, which was suppressed by specific short interfering (si)RNA knockdown of iPLA(2)-VIA. OGD was associated with an increase in iPLA(2)-VIA protein levels, whereas mRNA levels were unchanged. The levels of iPLA(2)-VIB mRNA and protein were not increased by OGD. RT-PCR and Western blot analysis identified a mouse iPLA(2)-VIA homolog to catalytically inactive 50-kDa iPLA(2)-VIA-ankyrin variants previously identified in humans. Both the mRNA and protein levels of this approximately 50-kDa variant were reduced significantly within 1 h following OGD. In C2C12 myoblasts, iPLA(2)-VIA seemed to predominantly reside at the endoplasmatic reticulum, where it accumulated further during OGD. A time-dependent reduction in cell viability during the early OGD period (3 h) was partially prevented by iPLA(2)-VIA knockdown or pharmacological inhibition (10 microM BEL), whereas iPLA(2)-VIA overexpression had no effect on cell viability. Taken together, these data demonstrate that OGD in C2C12 myotubes is associated with an increase in iPLA(2)-VIA activity that decreases cell viability. iPLA(2)-VIA activation may be modulated by changes in the levels of active and inactive iPLA(2)-VIA isoforms.

Protein phosphorylation/dephosphorylation and cytoskeletal reorganization regulate the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) during osmotic shrinkage; however, the mechanisms involved are unclear. We show that in cytoplasts, plasma membrane vesicles detached from Ehrlich ascites tumor cells (EATC) by cytochalasin treatment, NKCC1 activity evaluated as bumetanide-sensitive (86)Rb influx was increased compared with the basal level in intact cells yet could not be further increased by osmotic shrinkage. Accordingly, cytoplasts exhibited no regulatory volume increase after shrinkage. In cytoplasts, cortical F-actin organization was disrupted, and myosin II, which in shrunken EATC translocates to the cortical region, was absent. Moreover, NKCC1 activity was essentially insensitive to the myosin light chain kinase (MLCK) inhibitor ML-7, a potent blocker of shrinkage-induced NKCC1 activity in intact EATC. Cytoplast NKCC1 activity was potentiated by the Ser/Thr protein phosphatase inhibitor calyculin A, partially inhibited by the protein kinase A inhibitor H89, and blocked by the broad protein kinase inhibitor staurosporine. Cytoplasts exhibited increased protein levels of NKCC1, Ste20-related proline- and alanine-rich kinase (SPAK), and oxidative stress response kinase 1, yet they lacked the shrinkage-induced plasma membrane translocation of SPAK observed in intact cells. The basal phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was increased in cytoplasts compared with intact cells, yet in contrast to the substantial activation in shrunken intact cells, p38 MAPK could not be further activated by shrinkage of the cytoplasts. Together these findings indicate that shrinkage activation of NKCC1 in EATC is dependent on the cortical F-actin network, myosin II, and MLCK.

Osmotic swelling of NIH3T3 mouse fibroblasts activates a bromoenol lactone (BEL)-sensitive taurine efflux, pointing to the involvement of a Ca(2+)-independent phospholipase A(2) (iPLA(2)) (Lambert IH. J Membr Biol 192: 19-32, 2003). We report that taurine efflux from NIH3T3 cells was not only increased by cell swelling but also decreased by cell shrinkage. Arachidonic acid release to the cell exterior was similarly decreased by shrinkage yet not detectably increased by swelling. NIH3T3 cells were found to express cytosolic calcium-dependent cPLA(2)-IVA, cPLA(2)-IVB, cPLA(2)-IVC, iPLA(2)-VIA, iPLA(2)-VIB, and secretory sPLA(2)-V. Arachidonic acid release from swollen cells was partially inhibited by BEL and by the sPLA(2)-inhibitor manoalide. Cell swelling elicited BEL-sensitive arachidonic acid release from the nucleus, to which iPLA(2)-VIA localized. Exposure to the bee venom peptide melittin, to increase PLA(2) substrate availability, potentiated arachidonic acid release and osmolyte efflux in a volume-sensitive, 5-lipoxygenase-dependent, cyclooxygenase-independent manner. Melittin-induced arachidonic acid release was inhibited by manoalide and slightly but significantly by BEL. A BEL-sensitive, melittin-induced PLA(2) activity was also detected in lysates devoid of sPLA(2), indicating that both sPLA(2) and iPLA(2) contribute to arachidonic acid release in vivo. Swelling-induced taurine efflux was inhibited potently by BEL and partially by manoalide, whereas the reverse was true for melittin-induced taurine efflux. It is suggested that in NIH3T3 cells, swelling-induced taurine efflux is dependent at least in part on arachidonic acid release by iPLA(2) and possibly also by sPLA(2), whereas melittin-induced taurine efflux is dependent on arachidonic acid release by sPLA(2) and, to a lesser extent, iPLA(2).

The mechanisms controlling the volume-regulated anion current (VRAC) are incompletely elucidated. Here, we investigate the modulation of VRAC by cellular cholesterol and the potential involvement of F-actin, Rho, Rho kinase, and phosphatidylinositol-(4,5)-bisphosphate [PtdIns(4,5)P(2)] in this process. In Ehrlich-Lettre ascites (ELA) cells, a current with biophysical and pharmacological properties characteristic of VRAC was activated by hypotonic swelling. A 44% increase in cellular cholesterol content had no detectable effects on F-actin organization or VRAC activity. A 47% reduction in cellular cholesterol content increased cortical and stress fiber-associated F-actin content in swollen cells. Cholesterol depletion increased VRAC activation rate and maximal current after a modest (15%), but not after a severe (36%) reduction in extracellular osmolarity. The cholesterol depletion-induced increase in maximal VRAC current was prevented by F-actin disruption using latrunculin B (LB), while the current activation rate was unaffected by LB, but dependent on Rho kinase. Rho activity was decreased by approximately 20% in modestly, and approximately 50% in severely swollen cells. In modestly swollen cells, this reduction was prevented by cholesterol depletion, which also increased isotonic Rho activity. Thrombin, which stimulates Rho and causes actin polymerization, potentiated VRAC in modestly swollen cells. VRAC activity was unaffected by inclusion of a water-soluble PtdIns(4,5)P(2) analogue or a PtdIns(4,5)P(2)-blocking antibody in the pipette, or neomycin treatment to sequester PtdIns(4,5)P(2). It is suggested that in ELA cells, F-actin and Rho-Rho kinase modulate VRAC magnitude and activation rate, respectively, and that cholesterol depletion potentiates VRAC at least in part by preventing the hypotonicity-induced decrease in Rho activity and eliciting actin polymerization.

Maintenance of a stable cell volume and intracellular pH is critical for normal cell function. Arguably, two of the most important ion transporters involved in these processes are the Na+/H+ exchanger isoform 1 (NHE1) and Na+ -K+ -2Cl- cotransporter isoform 1 (NKCC1). Both NHE1 and NKCC1 are stimulated by cell shrinkage and by numerous other stimuli, including a wide range of hormones and growth factors, and for NHE1, intracellular acidification. Both transporters can be important regulators of cell volume, yet their activity also, directly or indirectly, affects the intracellular concentrations of Na+, Ca2+, Cl-, K+, and H+. Conversely, when either transporter responds to a stimulus other than cell shrinkage and when the driving force is directed to promote Na+ entry, one consequence may be cell swelling. Thus stimulation of NHE1 and/or NKCC1 by a deviation from homeostasis of a given parameter may regulate that parameter at the expense of compromising others, a coupling that may contribute to irreversible cell damage in a number of pathophysiological conditions. This review addresses the roles of NHE1 and NKCC1 in the cellular responses to physiological and pathophysiological stress. The aim is to provide a comprehensive overview of the mechanisms and consequences of stress-induced stimulation of these transporters with focus on the heart, brain, and blood. The physiological stressors reviewed are metabolic/exercise stress, osmotic stress, and mechanical stress, conditions in which NHE1 and NKCC1 play important physiological roles. With respect to pathophysiology, the focus is on ischemia and severe hypoxia where the roles of NHE1 and NKCC1 have been widely studied yet remain controversial and incompletely elucidated.

In this report, we describe the cloning, cellular localization, and functional characteristics of Na(+)/H(+) exchanger 1 (NHE1) from red blood cells of the winter flounder Pseudopleuronectes americanus (paNHE1). The paNHE1 protein localizes primarily to the marginal band and exhibits a 74% similarity to the trout beta-NHE, and 65% to the human NHE1 (hNHE1). Functionally, paNHE1 shares characteristics of both beta-NHE and hNHE1 in that it is activated both by manipulations that increase cAMP and by cell shrinkage, respectively. In accordance, the paNHE1 protein exhibits both protein kinase A consensus sites as in beta-NHE and a region of high homology to that required for shrinkage-dependent activation of hNHE1. After shrinkage-dependent activation of paNHE1 and resulting activation of a Cl(-)/HCO(3)(-) exchanger, their parallel operation results in net uptake of NaCl and osmotically obliged water. Activation of paNHE1 by cAMP is at least additive to that elicited by osmotic shrinkage, suggesting that these stimuli regulate paNHE1 by distinct mechanisms. Finally, exposure to the serine/threonine phosphatase inhibitor calyculin A potently activates paNHE1, and this activation is also additive to that induced by shrinkage or cAMP.

Arachidonic acid or its metabolites have been implicated in the regulatory volume decrease (RVD) response after hypotonic cell swelling in some mammalian cells. The present study investigated the role of arachidonic acid (AA) during RVD in the human neuroblastoma cell line CHP-100. During the first nine minutes of hypo-osmotic exposure the rate of 3H-arachidonic acid (3H-AA) release increased to 250 +/- 19% (mean +/- SE, n = 22) as compared with cells under iso-osmotic conditions. This release was significantly inhibited after preincubation with AACOCF3, an inhibitor of the 85-kDa cytosolic phospholipase A2 (cPLA2). This indicates that a PLA2, most likely the 85-kDa cPLA2 is activated during cell swelling. In contrast, preincubation with U73122, an inhibitor of phospholipase C, did not affect the swelling-induced release of 3H-AA. Swelling-activated efflux of 36Cl and 3H-taurine were inhibited after preincubation with AACOCF3. Thus the swelling-induced activation of cPLA2 may be essential for stimulation of both 36Cl and 3H-taurine efflux during RVD. As the above observation could result from a direct effect of AA or its metabolite leukotriene D4 (LTD4), the effects of these agents were investigated on swelling-induced 36Cl and 3H-taurine effluxes. In the presence of high concentrations of extracellular AA, the swelling-induced efflux of 36Cl and 3H-taurine were inhibited significantly. In contrast, addition of exogenous LTD4 had no significant effect on the swelling-activated 36Cl efflux. Furthermore, exogenous AA increased cytosolic calcium levels as measured in single cells loaded with the calcium sensitive dye Fura-2. On the basis of these results we propose that cell swelling activates phospholipase A2 and that this activation via an increased production of AA or some AA metabolite(s) other than LTD4 is essential for RVD.