Intracellular Carbonic Anhydrase Activity Sensitizes Cancer Cell pH Signaling to Dynamic Changes in CO₂ Partial Pressure

Background: Intracellular carbonic anhydrase (CA_i) activity, commonly detected in cancer, accelerates CO₂/HCO₃⁻ equilibration.

Results: In cells with high CA_i activity, fluctuations in pCO₂ (which can arise from intermittent blood flow) evoke substantial intracellular pH oscillations that modulate downstream pathways (e.g., calcium, mTOR).

Conclusions: CA_i transduces the state of perfusion to intracellular signaling.

Significance: Coupling between environment and cell behavior may influence cancer progression.

Abstract

Carbonic anhydrase (CA) enzymes catalyze the chemical equilibration among CO₂, HCO₃⁻ and H⁺. Intracellular CA (CA_i) isoforms are present in certain types of cancer, and growing evidence suggests that low levels correlate with disease severity. However, their physiological role remains unclear. Cancer cell CA_i activity, measured as cytoplasmic CO₂ hydration rate (k_f), ranged from high in colorectal HCT116 (∼2 s⁻¹), bladder RT112 and colorectal HT29, moderate in fibrosarcoma HT1080 to negligible (i.e. spontaneous k_f = 0.18 s⁻¹) in cervical HeLa and breast MDA-MB-468 cells. CA_i activity in cells correlated with CAII immunoreactivity and enzymatic activity in membrane-free lysates, suggesting that soluble CAII is an important intracellular isoform. CA_i catalysis was not obligatory for supporting acid extrusion by H⁺ efflux or HCO₃⁻ influx, nor for maintaining intracellular pH (pH_i) uniformity. However, in the absence of CA_i activity, acid loading from a highly alkaline pH_i was rate-limited by HCO₃⁻ supply from spontaneous CO₂ hydration. In solid tumors, time-dependence of blood flow can result in fluctuations of CO₂ partial pressure (pCO₂) that disturb cytoplasmic CO₂-HCO₃⁻-H⁺ equilibrium. In cancer cells with high CA_i activity, extracellular pCO₂ fluctuations evoked faster and larger pH_i oscillations. Functionally, these resulted in larger pH-dependent intracellular [Ca²⁺] oscillations and stronger inhibition of the mTORC1 pathway reported by S6 kinase phosphorylation. In contrast, the pH_i of cells with low CA_i activity was less responsive to pCO₂ fluctuations. Such low pass filtering would “buffer” cancer cell pH_i from non-steady-state extracellular pCO₂. Thus, CA_i activity determines the coupling between pCO₂ (a function of tumor perfusion) and pH_i (a potent modulator of cancer cell physiology).

Introduction

The chemical components of carbonic buffer (CO₂/HCO₃⁻) participate in metabolic reactions and in pH homeostasis (1–3). Due to rapid permeation of CO₂ gas across cell membranes (4), carbonic buffer is present in both intra- and extracellular tissue compartments. On the time scale of cellular physiology, the spontaneous equilibration of carbonic buffer is relatively slow, as exemplified by a CO₂ hydration time constant of several seconds (rate constant k_f = 0.18 s⁻¹) (5). Consequently, biological processes that involve a change in the concentration of CO₂, HCO₃⁻ or H⁺ can become rate-limited by carbonic buffer re-equilibration. This limitation has presumably driven the evolution of at least a dozen mammalian carbonic anhydrase (CA)² isozymes that accelerate CO₂/HCO₃⁻ equilibration (6, 7).

The CAs are grouped as intra- (CA_i) or extracellular (CA_e) depending on the orientation of the catalytic site (5–8). Activity assays and immunotechniques have identified CA_i and CA_e isoforms in cancer cells (9–17). Physiologically, CA_e isoforms, such as CAIX and CAXII, facilitate CO₂ and H⁺ diffusion across the continuous and tortuous interstitial space (18, 19). Thus, CA_e activity can improve the venting of acidic products of metabolism over the long diffusion distances found in inadequately perfused solid tumors, allowing their faster growth (20, 21). The role of CA_i isoforms in cancer physiology is still debated. Down-regulation of gap junctions in cancer cells prevents the intracellular compartment from becoming syncytial (22), and this restricts the spatial range over which CA_i activity could facilitate CO₂ or H⁺ diffusion. Previously, it has been suggested that CA_i activity facilitates the transport of HCO₃⁻ or H⁺ ions across membranes by reducing the extent to which cytoplasmic reactions slow the delivery or removal of the transported ion (23–26). To benefit from CA activity, these transporter-evoked H⁺ or HCO₃⁻ fluxes would have to exceed the spontaneous chemical re-equilibration kinetics of carbonic buffer (27).

Another source of disturbance to carbonic buffer equilibrium is fluctuating CO₂ partial pressure (pCO₂). Cancer cells produce large quantities of CO₂ from titration of acids (e.g. lactic) with HCO₃⁻ and decarboxylation by mitochondria and the pentose phosphate shunt (28). Ultimately, the excess CO₂ must be removed with the blood flow. In tumors, vasomotion and hemodynamic factors can produce cycles of intermittent blood flow, commonly observed with periodicities of several minutes (29–33). Unstable perfusion is the basis for acute hypoxia (32, 34–40), characterized by oxygenation-reoxygenation cycles as fast as 2/min (30) and amplitudes of tens of mmHg O₂ (30, 35, 41, 42). Episodes of inadequate blood flow produce “closed” pockets of blood, which become oxygen-depleted and accumulate CO₂ (43–45). Periodic restoration of flow returns pCO₂ and pO₂ to normal. The resulting fluctuations in pCO₂ (mirroring pO₂) are transmitted across the intra- and extracellular compartments because CO₂ gas (unlike H⁺ or HCO₃⁻ ions) crosses membranes rapidly (4). Due to reversible intracellular CO₂ hydration, pH_i will respond to pCO₂ fluctuations, but the coupling between these depends critically on CA_i activity. Because cell behavior is strongly modulated by H⁺ ions (46–48), CA_i activity may act as an important transducer between blood flow and cell signaling.

The aim of the present study was to measure CA_i activity in the native environment of intact cancer cells and to identify the physiological processes, relevant to cancer, that depend on CA_i activity. We describe a novel role for CA_i activity in sensitizing cancer cell pH to pCO₂ changes, thereby linking metabolism and perfusion with pH_i-responsive signaling cascades.

EXPERIMENTAL PROCEDURES

Cell Lines and Cell Culture

Cancer cell lines were obtained from ATCC (colorectal HCT116), Cancer Research UK (bladder RT112, colorectal HT29, fibrosarcomal HT1080, and breast MDA-MB-468) or were a gift from Professor Silvia Pastorekova, Bratislava, Slovakia (cervical HeLa). Fibroblast cell lines were obtained from Lonza (NHDF-Ad adult dermal fibroblasts and InMyoFib intestinal myofibroblasts) or ATCC (normal colon CCD-112-Con and carcinoma-derived Hs675.T fibroblasts). Cells were grown in HCO₃⁻-containing DMEM in 5% CO₂ and 21% O₂ for 48 h until 80–100% confluent (Galaxy 48R, New Brunswick). For some experiments, cells were cultured in the presence of 1 mm dimethyl oxalylglycine (DMOG) dissolved in DMSO.

CAII Knockdown

CA2 gene expression in HCT116 cells was silenced using one of four shRNA constructs cloned into psi-LVRU6-GFP lentiviral vector targeting the 351st, 493rd, 597th, and 695th position of CA2 mRNA (NCBI Reference Sequence NM_000067). All constructs, including scrambled-eGFP, were purchased from Genecopoiea. Stable HCT116 cell clones with silenced CA2 gene expression were selected in the presence of 2 μg/ml puromycin for 2 weeks, and selected clones were pooled together for experiments.

Western Blotting

Cells were lysed at 4 °C (1% Triton X-100, 0.5% Nonidet P-40 substitute (Applichem), 150 mm NaCl, 50 mm NaF, 50 mm Tris-HCl (pH 7.5) plus Roche Applied Science protease inhibitor mixture) and pelleted at 13,000 rpm for 20 min at 4 °C. Proteins were resolved by SDS-PAGE, transferred to a PVDF membrane (Bio-Rad), and detected using mouse monoclonal M75 antibody against human CAIX (a gift from Prof. Pastorekova; Ref. 49); antibodies against CAI, -III (R&D Systems), -II (Novus), -VII (AbD Serotech), and -XIII (Abcam); and goat polyclonal antibody to actin (Santa Cruz Biotechnology). Kinase phosphorylation in cells cultured to high density was measured with the phospho-MAPK array profiler (R&D Systems) and mechanistic target of rapamycin (mTOR) signaling kit (Cell Signaling Technology).

Measuring Carbonic Anhydrase Activity in Lysates

Cells were lysed by repeated freeze-thaw cycles in buffer containing 140 mm potassium gluconate, 0.5 mm EGTA, 1 mm MgCl₂, 15 mm Hepes, 15 mm Mes at pH 7.8 (4 °C), and protease inhibitor. Membranes were removed by centrifugation (20 min at 15,000 rpm at 4 °C), and the supernatant was diluted to a total protein concentration between 1 and 10 mg/ml (Bradford assay). The CA-catalyzed reaction was triggered by adding 0.33 ml CO₂-saturated water to 0.67 ml of lysate in a stirred chamber at 4 °C. The time course of pH (Hamilton Biotrode) was fitted with a kinetic model (19) to obtain the CO₂ hydration rate constant k_f.

Superfusion

CO₂/HCO₃⁻-free solution contained 125 mm NaCl, 20 mm Hepes (pH to 7.4 with NaOH), 4.5 mm KCl, 11 mm glucose, 1 mm CaCl₂, and 1 mm MgCl₂. For CO₂/HCO₃⁻-buffered solution, Hepes was replaced with 22 mm NaHCO₃, and the solution was bubbled with either 5% CO₂ (pH 7.4) or 20% (pH 6.8). For Cl⁻-free solutions, Cl⁻ was replaced with gluconate. For acetate-containing solution, 80 mm NaCl was replaced with 80 mm sodium acetate. For NH₄⁺-containing solution, 20 mm NaCl was replaced with 20 mm NH₄Cl. Cells were superfused (37 °C at 4 ml/min) in a poly-l-lysine-treated Perspex chamber (solution exchange time constant <3 s) except for fluctuating pCO₂ protocols, which were performed in 2-ml Lab-Tek chambers (Nunc) that allow a more gradual solution exchange.

Fluorescence Measurements of Intracellular pH and Calcium

Cells were imaged confocally using a Zeiss LSM 700 confocal system on an Axiovert inverted microscope. To measure pH_i, cells were AM-loaded for 10 min with 10 μm cSNARF1 (excitation, 555 nm; emission, 580 and 640 nm). The fluorescence ratio was converted to pH_i using a calibration curve obtained by the nigericin method (50). Calibration was performed twice a year for each cell line, and the stability of the curve was tested by the null point method (51). Membrane H⁺ (or H⁺-equivalent) flux was calculated as the rate of pH_i change × buffering capacity (3). Intrinsic buffering capacity was measured in separate experiments using the graded ammonium removal technique (3). CO₂/HCO₃⁻-dependent buffering was estimated using the equation for an open buffer system: 2.303 × [HCO₃⁻]_i. Intracellular [HCO₃⁻] was calculated by best fitting a mathematical model of pH_i regulation to the time course of pH_i recovery from an acid or base load (see below). This approach derives instantaneous carbonic buffering capacity (rather than assuming that the buffer is distributed at equilibrium across the cell membrane at all times). To measure [Ca²⁺]_i, cells were AM-loaded for 20 min with 50 μm Fluo3 (excitation, 488 nm; emission, >510 nm). For co-cultures, regions with distinct areas of cancer cells and fibroblasts (distinguished by cell morphology and size) were selected for imaging.

Mathematical Modeling

A mathematical model of pH_i control was parameterized using data for buffering capacity (β) and membrane transport fluxes of H⁺ and HCO₃⁻ ions (J^H and J^HCO3) (3). The carbonic buffer equilibrium constant, K_CO2, was 10^−6.1 m, and the spontaneous CO₂ hydration rate constant (k_f) was 0.18 s⁻¹ (the reverse rate constant, k_r, was k_f/K_CO2). CA_i activity was represented as a dimensionless scalar (ca). Surface area/volume ratio was 1/h where h is the monolayer height (10 μm). CO₂ permeability, p, was 10⁴ μm/s (4). pCO₂ was a sinusoidal function, f, of amplitude A, frequency φ, and baseline of 5% CO₂. The equations are as follows.

RESULTS

CA_i Activity in Cancer and Fibroblast Cell Lines

Total CA_i activity was measured in intact cells superfused continuously with physiological salt solution at body temperature. The CA_i-catalyzed reaction was triggered by switching between CO₂/HCO₃⁻-free and 5% CO₂/22 mm HCO₃⁻-buffered superfusates. The time constant of solution exchange (2.7 s; determined by fluorescently labeling one solution with 30 μm fluorescein) was adequately fast to ensure that pCO₂ was manipulated rapidly. CA_i-catalyzed reaction kinetics were determined from the initial rate of pH_i change (reported using the fluorescent pH indicator cSNARF1 AM-loaded into cells) and intrinsic buffering capacity (52). Fig. 1A, panel i, shows an example pH_i time course recorded from colorectal HCT116 cells paired with an experiment performed in the presence of the broad spectrum CA inhibitor acetazolamide (ATZ; 100 μm) to determine spontaneous kinetics over a matching pH_i range. CA_i activity was expressed relative to spontaneous reaction kinetics (Fig. 1A, panel ii). Experiments were also performed on bladder RT112, colorectal HT29, fibrosarcoma HT1080, cervical HeLa, and breast MDA-MB-468 cells as well as dermal NHDF-Ad, colonic CCD-112-CoN fibroblasts, and intestinal InMyoFib myofibroblasts. CA_i activity ranged from ∼9-fold above the spontaneous rate in HCT116 cells to very low in HeLa, MDA-MB-468, and fibroblast/fibroblast-related cells. Thus, the CO₂ hydration capacity of cancer cell cytoplasm spanned over an order of magnitude in range and was cell line-dependent.

FIGURE 1.

Intracellular carbonic anhydrase activity. A, panel i, determination of cytoplasmic CO₂ hydration rate from pH_i dynamics in intact HCT116 cells (AM-loaded with pH dye cSNARF1). Switching between CO₂/HCO₃⁻-free and 5% CO₂, 22 mm HCO₃⁻-containing superfusate triggers intracellular CO₂ hydration. The experiment was repeated with 100 μm ATZ to inhibit CA. Inset, pH_i response to CO₂ addition. Panel ii, CA_i activity in cancer and fibroblast/fibroblast-related cells (n > 20 each), normalized to measurements in the presence of ATZ (k_f = 0.18 s⁻¹). B, panel i, measuring CA activity in membrane-free RT112 lysates. Panel ii, CA_s activity normalized to spontaneous rate determined in ATZ (expressed per mg/ml of total protein). C, panel i, knockdown of CAII in HCT116 cells using one of four shRNA constructs, scrambled shRNA (sham) and non-transfected wild type (WT). *, p < 0.05, relative to WT. Panel ii, total CA_i activity in knockdown, sham, and WT HCT116 cells (n > 20 each). Panel iii, Western blot for soluble CA isoforms other than CAII in HCT116 cells treated with shRNA. D, Western blot for CAII and CAIX under control (normoxic) incubation or 48 h in 1 mm dimethyl oxalylglycine (DMOG). Error bars represent S.E.

Soluble cytoplasmic CA (CA_s) isoforms (e.g. CAI, -II, -III, -VII, and -XIII) are plausible contributors to CA_i activity. This was tested in cell lysates after removing membranes by centrifugation. Lysates were injected with CO₂-saturated water, and the CO₂ hydration constant was estimated from the time course of medium pH at 4 °C (Fig. 1B, panel i). CA_s activity was quantified in terms of the CO₂ hydration rate constant relative to the measurement in the presence of ATZ (100 μm). Kinetic data were normalized to total protein concentration (19). Substantial CA_s catalysis was detected in lysates of cancer cells with high total CA_i activity determined in intact cells. This argues that soluble isoforms underlie at least part of intracellular CO₂ hydration catalysis (Fig. 1B, panel ii). The contribution of CAII to total CA_i activity was tested in HCT116 cells by knockdown using shRNA constructs 351, 495, 597 and 695 and compared with scrambled shRNA and wild-type cells. Construct 695 resulted in the most substantial decrease in CAII immunoreactivity (Fig. 1C, panel i), and it reduced total CA_i activity by ∼90% relative to wild-type (Fig. 1C, panel ii). To test whether the decrease in total CA_i activity is attributable to disruption of CAII alone, the specificity of shRNA constructs 695 and 597 was investigated. Constructs 695 and 597 have very low sequence homology (16–42%) with membrane-associated CAs (IV, IX, XII, and XIV) and low homology (53–68%) with soluble isoforms other than CAII (I, III, VII, and XIII). Of the soluble isoforms, HCT116 expressed only low levels of CAVII and -XIII, and immunoreactivity for CAI and -III was absent (Fig. 1C, panel iii). Constructs 695 and 597 did not alter this expression pattern, indicating that the knockdown experiment reduced CA_i activity by targeting CAII selectively. The findings indicate that CAII is a principal contributor to CA_i activity in HCT116 cells.

Across the tested cancer, fibroblast, and fibroblast-related cells, CAII immunoreactivity (Fig. 1D) correlated with in situ CA_i activity (Pearson's correlation coefficient r² = 0.83). CAII expression was notably absent in fibroblast and fibroblast-related cells, including cancer-derived Hs675.T cells. In cancer cells, CAII expression correlated strongly (r² = 0.97) with CA_s activity measured in membrane-free lysates. As expected from active site topology, the expression of membrane-tethered CAIX did not correlate with CA_i activity. Hypoxia plays an important role in cancer biology by regulating gene expression through mechanisms that include hypoxia-inducible factor. To investigate the effect of long term hypoxia on CAII expression, Western blot analysis was performed on cells incubated for 48 h with the hypoxia-inducible factor-stabilizing drug dimethyl oxalylglycine (3, 53). This treatment did not affect CAII expression but produced the expected induction of CAIX in RT112, HT1080, MDA-MB-468, and HeLa (54) and to a lesser degree in NHDF-Ad and CCD-112-CoN fibroblasts.

Role of CA_i Activity in pH_i Regulation

The supply of H⁺ or HCO₃⁻ ions for pH_i-regulatory transport across membranes may be rate-limited by the CA_i-catalyzed reaction. This was investigated in RT112, MDA-MB-468, and HCT116 cells.

RT112 cells have naturally high CA_i activity and can produce large HCO₃⁻ fluxes at both low and high pH_i (3). Acid extrusion was evoked at low pH_i by means of an “ammonium prepulse” solution maneuver performed on cSNARF1-loaded cells (Fig. 2A, panel i). An “acetate prepulse” was performed to raise pH_i. Because abrupt base loading of cytoplasm upon acetate removal drives carbonic buffer out-of-equilibrium, cells were superfused briefly in Cl⁻-free medium to allow buffer re-equilibration (55). Subsequent re-exposure to Cl⁻-containing superfusates activated acid loading transporters (Fig. 2A, panel ii). H⁺/H⁺-equivalent flux was calculated as the product of the rate of pH_i change and buffering capacity (the carbonic buffer component was estimated using a mathematical model to predict out-of-equilibrium behavior during dynamic pH_i regulation). pH_i-regulatory fluxes, shown in Fig. 2A, panel iii, were symmetrical around the resting pH_i. To establish the contribution of Na⁺/H⁺ exchange (NHE) to pH_i regulation, measurements were repeated in the presence of 30 μm 5-(N,N-dimethyl)amiloride (DMA). Acid loading was DMA-insensitive, whereas acid extrusion flux was reduced only modestly, confirming a minor role for NHE in pH_i regulation in RT112 cells (Fig. 2A, panel iii, inset). Replacing superfusate carbonic buffer with Hepes reduced flux substantially, indicating that pH_i regulation relies principally on HCO₃⁻ transport. The effect of CA_i activity on acid/base membrane transport was inferred from the effect of 100 μm ATZ (in the presence of carbonic buffer). Although ATZ targets both intra- and extracellular CAs, the latter isoforms would not be exerting a net catalytic effect because superfusion presents cells with pre-equilibrated buffer at all times (19). ATZ did not affect resting pH_i or acid extrusion flux, but it limited acid-loading fluxes to no greater than ∼14 mm/min. This flux is equal to the maximal rate of HCO₃⁻ delivery by CO₂ hydration under spontaneous reaction kinetics (k_f × [CO₂] = 14 mm/min). Thus, acid loading by means of HCO₃⁻ export can be rate-limited by CA_i activity, but this requires large fluxes evoked by substantially raised pH_i.

FIGURE 2.

Role of CA_i activity in membrane H⁺/HCO₃⁻ transport and cytoplasmic H⁺ diffusion. A, 20 mm ammonium (panel i) and 80 mm acetate (panel ii) prepulse performed on RT112 cells (5% CO₂/22 mm HCO₃⁻ superfusate). Experiment repeated in 100 μm ATZ (dotted line). Following acetate removal, cells were stabilized in Cl⁻-free superfusate to allow for CO₂/HCO₃⁻ re-equilibration. Panel iii, acid-extrusion and acid-loading fluxes (n > 20). Dashed line, limit of spontaneous production of HCO₃⁻. Inset, effect of 30 μm DMA. B, 20 mm ammonium prepulse performed on MDA-MB-468 cells in the absence (panel i) or presence (panel ii) of 5% CO₂/22 mm HCO₃⁻. Cytoplasmic pH was measured in concentric layers of thickness ∼1.5 μm near surface membrane (black trace) and at cell core (gray trace). Mean of 10 cells (error bars not shown). C, panel i, 20 mm ammonium prepulse performed on HCT116 cells in the absence of CO₂/HCO₃⁻. Panel ii, intrinsic buffering capacity (measured by graded ammonium removal) in control HCT116 cells and cells cultured with 6 mm carnosine for 48 h (n > 20). Panel iii, NHE flux (n > 20) in control (CO₂/HCO₃⁻-free), in carnosine-loaded cells (CO₂/HCO₃⁻-free), and in the presence of 5% CO₂/22 mm HCO₃⁻ (determined by subtracting the DMA-sensitive component of flux). Panel iv, H⁺-equivalent flux in CO₂/HCO₃⁻ buffer measured in CAII knockdown HCT116 cells (construct 695) and compared with scrambled (sham). Experiments were repeated in DMA to inhibit the NHE component of acid extrusion (n > 15). Error bars represent S.E.

Intracellular H⁺ ions diffuse considerably slower than expected from their low atomic weight because of extensive binding to larger buffer molecules, including immobile proteins (56, 57). NHE activity could plausibly be rate-limited by a slow diffusive supply of H⁺ ions and therefore require CA_i activity for delivering H⁺ ions from CO₂ hydration. As shown in non-cancerous cells, CA_i activity can facilitate H⁺ diffusion by improving the reaction kinetics of CO₂/HCO₃⁻, a mobile buffer (58, 59). If the intrinsic H⁺ ion diffusivity in cytoplasm were sufficiently restricted, a large acid/base flux at the surface membrane would be expected to produce measurable pH_i non-uniformity in the absence of carbonic buffer. This was tested in MDA-MB-468 cells, which produce very high NHE fluxes at low pH_i (3). Cells were trypsinized to produce spherical cells with the smallest possible surface area-to-volume ratio, i.e. longest average surface-to-core diffusion distance. The mean radius of MDA-MB-468 cells was 7.11 ± 0.12 μm, which is typical of most cancer cells. NHE is functional in the presence and absence of carbonic buffer; therefore it is possible to investigate the effect of removing CO₂/HCO₃⁻ on spatial H⁺ dynamics in cytoplasm. pH_i uniformity during rapid acid extrusion was assessed by imaging pH_i confocally near the surface membrane and at the core of the cell. The pH_i time courses shown in Fig. 2B, panel i, indicate that even at maximal acid extrusion rates, pH_i remained uniform in the absence of carbonic buffer. The addition of carbonic buffer did not affect the degree of pH_i uniformity (Fig. 2B, panel ii). Thus, carbonic buffer and hence CA_i activity are not required for ensuring adequate H⁺ diffusion in MDA-MB-468 cells. These findings are indicative of good diffusive coupling by intrinsic H⁺ buffers.

Measurements of pH_i gradients in bulk cytoplasm may not identify diffusion barriers in the immediate environment of the transport protein. An example of such a barrier is slow H⁺ transfer from immobile buffers to the transporter proteins (60, 61). Given that CO₂ is a highly penetrating source of H⁺ ions (26), a role for CA_i activity in overcoming these localized H⁺ barriers is conceivable. To test for possible submembrane barriers, acid extrusion was measured in HCT116 cells, which have naturally high CA_i activity and the capacity to produce large NHE fluxes (3). If CA_i activity were important for delivering H⁺ ions to NHE protein, then removal of carbonic buffer would slow overall flux to a baseline set by the intrinsic capacity of the cytoplasm to supply the transporter with H⁺ ions. Loading cytoplasm with exogenous mobile buffers, such as imidazoles, would then be expected to rescue acid extrusion (60). Fig. 2C, panel i, shows the time course of pH_i recovery from low pH_i in the absence of CO₂/HCO₃⁻. This eliminates CO₂-sourced delivery of H⁺ ions and would expose any submembrane diffusional barriers. Experiments were repeated on cells loaded with the imidazole-containing mobile buffer carnosine (6 mm for 48 h). Intrinsic buffering capacity, measured by the “graded ammonium removal” technique (62), was higher in carnosine-incubated cells, consistent with the loading of cytoplasm with an exogenous buffer of pK 6.7 (similar to that of carnosine) and concentration of 12 mm (Fig. 2C, panel ii). After accounting for the additional buffering capacity, flux in carnosine-loaded cells was no different from control (Fig. 2C, panel iii). Further experiments were performed on control HCT116 cells superfused with carbonic buffer. Because HCO₃⁻-dependent mechanisms (e.g. Na⁺-HCO₃⁻ co-transport) are activated in the presence of carbonic buffer, NHE-mediated flux was calculated by subtracting the DMA-sensitive component from the total flux. Neither CO₂/HCO₃⁻ nor carnosine increased NHE flux (Fig. 2C, panel iii), arguing that these additional mobile buffers are not required for delivering H⁺ ions to NHE. Thus, CA_i catalysis is unlikely to accelerate transport by providing an additional route of H⁺ delivery. An earlier study had suggested that imidazole groups projecting from the CAII molecule deliver H⁺ ions to H⁺ transporters without involving the catalytic process (60). Because large acid extrusion fluxes could be produced by CAII-negative MDA-MB-468 cells, an acatalytic effect of CAII is unlikely to be an absolute requirement for fast NHE activity. This was explored further in CAII knockdown HCT116 cells (construct 695; Fig. 1C, panel i). Acid extrusion in carbonic buffer, which includes NHE and HCO₃⁻-dependent components, was not different in CAII-negative cells compared with scrambled controls (Fig. 2C, panel iv). The HCO₃⁻-dependent flux component, measured by inhibiting NHE with DMA, was also unaffected by CAII knockdown (Fig. 2C, panel iv).

Role of CA_i Activity during Fluctuations in pCO₂

To explore the effects of CA_i activity during pCO₂ fluctuations associated with intermittent blood flow, superfusion of confluent monolayers (grown in slow exchange chambers) was alternated between 5% CO₂/22 mm HCO₃⁻ (pH 7.4; representing normal arterial blood plasma) and 20% CO₂/22 mm HCO₃⁻ (pH 6.8; representing respiratory acidosis) with a periodicity of 4 min. By labeling one solution with fluorescein (30 μm) in separate experiments, the solution mixing interval was estimated to be ∼15 s, which allows for a relatively slow transition between 5 and 20% CO₂. Fig. 3A shows the time course of extracellular [H⁺] in the superfusion chamber reported by fluorescein (an indicator of solution pH) added to both solutions at 30 μm. The response of intracellular [H⁺] ([H⁺]_i) to pCO₂ fluctuations was recorded in cSNARF1-loaded monolayers of cells with high CA_i activity (HCT116 and HT29; Fig. 3, B and C), low CA_i activity (HeLa and MDA-MB-468; Fig. 3, D and E), and CAII knockdown HCT116 (CAII-KD). The measured [H⁺]_i fluctuations were quantified in terms of root mean square in Fig. 3F. Experiments were repeated in the presence of ATZ (100 μm) to inhibit CA activity. The temporal behavior of [H⁺]_i was ATZ-sensitive in HCT116 and HT29 cells but not in HeLa, MDA-MB-468, or CAII knockdown HCT116 cells (Fig. 3F). These responses did not correlate with normoxic CAIX expression (high in MDA-MB-468 and HT29 and low in HeLa and HCT116), and therefore cannot be attributed to the inhibition of exofacial CA isoforms. Thus, in cells with high CA_i activity, [H⁺]_i fluctuated faster and over a larger range, demonstrating a tighter coupling between pCO₂ and cytoplasmic [H⁺].

FIGURE 3.

Effect of CA_i activity on cytoplasmic pH dynamics during fluctuating pCO₂. pCO₂ was changed between 5 and 20%. A, extracellular pH reported using fluorescein (30 μm). B, intracellular [H⁺] measured in cSNARF1-loaded HCT116 monolayers with naturally high CA_i activity. The protocol was repeated in 100 μm ATZ. The rate of change, d[H⁺]/dt, is plotted below the [H⁺] time course. An average of >20 cells was analyzed. Experiments were also performed on HT29 monolayers with naturally high CA_i activity (C), HeLa (D), and MDA-MB-468 monolayers with naturally low CA_i activity (E). F, intracellular [H⁺] changes quantified as root mean square (RMS). *, p < 0.05, relative to control. G, simulated sinusoidal waveform of pCO₂ and its effect on intracellular [H⁺]. H, model simulation of the effect of CA activity (10-fold acceleration of hydration) on peak-to-nadir [H⁺]_i amplitude (percentage increase). Error bars represent S.E.

The relationship between pCO₂ and [H⁺]_i was explored further using a mathematical model parameterized using data for CA_i activity and buffering capacity in HCT116 cells (3). Smooth transitions between normal and raised pCO₂ were simulated with a sinusoidal wave (Fig. 3G) over a range of periodicity and amplitude. Fig. 3H shows the predicted effect of 10-fold CA_i activity on the size of [H⁺]_i fluctuations. [H⁺]_i fluctuations were amplified 30% for periodicities of 4 min and at least doubled for periodicities of <2 min. The relative effect of CA_i activity on [H⁺]_i dynamics was essentially independent of pCO₂ amplitude. pH_i-regulatory H⁺ and HCO₃⁻ membrane transport is expected to curtail the extent of [H⁺]_i changes, but mathematical modeling (not shown) predicts this to be a minor effect because the magnitude of membrane H⁺/HCO₃⁻ transport is considerably smaller than that of CO₂ flux, particularly for short pCO₂ wave periodicities.

In summary, CA_i activity sensitizes cancer cell pH to fluctuations in pCO₂. In contrast, cytoplasm with low CA_i activity behaves as a low pass filter that dampens [H⁺]_i changes. Essentially all biological processes are pH-sensitive; therefore the effect that CA_i activity has on [H⁺]_i dynamics during pCO₂ fluctuations is potentially of major functional importance. This was explored by measuring Ca²⁺ dynamics and kinase activity as examples of intracellular signaling.

Ca²⁺ Signaling

The behavior of the signaling ion Ca²⁺ was measured in Fluo3-loaded HCT116 cells during oscillations of pCO₂ between 5 and 20% (4-min periodicity). A significant increase in Fluo3 fluorescence was observed upon returning pCO₂ from 20 to 5% (Fig. 4A). Fluo3 fluorescence is modestly pH-sensitive, and to assess the magnitude of this artifact, HCT116 cells superfused in Ca²⁺-free and CO₂/HCO₃⁻-free media were exposed to 40 mm acetate for 4 min to evoke a pH_i change comparable to that caused by the pCO₂ fluctuations. Fluo3 fluorescence did not change substantially during this protocol (Fig. 4B), arguing that the responses shown in Fig. 4A report a genuine rise of cytoplasmic [Ca²⁺] ([Ca²⁺]_i). The mechanism of this response was explored using inhibitor drugs and Ca²⁺ buffers (Fig. 4C). The Ca²⁺ response was dampened in cells AM-loaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA; 100 μm), adding to the evidence that Fluo3 reports [Ca²⁺]_i changes rather than pH_i. Responses were considerably greater when intracellular Ca²⁺ stores were depleted by pretreatment with 10 μm thapsigargin (sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitor). In the absence of extracellular Ca²⁺ (nominally Ca²⁺-free; residual Ca²⁺ buffered with 1 mm EGTA), the Fluo3 response was abolished, arguing that Ca²⁺ influx is required. Collectively, these data suggest that cytoplasmic alkalinization evokes Ca²⁺ influx through store-operated channels, such as Orai (64). In support of this, 2-aminoethoxydiphenyl borate (100 μm) blocked the Ca²⁺ response. The CO₂-evoked [Ca²⁺]_i response was dampened in the presence of 100 μm ATZ, illustrating a role for CA_i activity in accentuating a cellular response to changing pCO₂. Experiments on co-cultures of HCT116 cells with fibroblasts of low CA_i activity (NHDF-Ad, CCD-112-CoN, InMyoFib; Fig. 4, D–F) confirmed that CA_i activity can influence the Ca²⁺ response to pCO₂ fluctuations in a cell-dependent manner.

FIGURE 4.

Effect of CA_i-mediated CO₂-pH coupling on Ca²⁺ signaling. A, intracellular Ca²⁺ (Fluo3 F/F₀ ratio) during 5–20% CO₂ oscillations (cycle length of 4 min). B, transient pH_i change evoked by acetate (in the absence of extracellular Ca²⁺ and absence of CO₂/HCO₃⁻) did not evoke a Fluo3 response. C, Fluo3 response recorded once consistent cyclic behavior had been attained. The response was reduced in 100 μm ATZ or in cells AM-loaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) (Ca²⁺ buffer; 100 μm), abolished by removing extracellular Ca²⁺ (replaced with EGTA Ca²⁺ buffer) or in 100 μm 2-aminoethoxydiphenyl borate (2-APB), and augmented in 10 μm thapsigargin (TG)-pretreated cells (endoplasmic reticulum depletion). Data are the means of >50 cells. HCT116 cancer cells co-cultured with either CCD112-CoN fibroblasts (D), InMyoFib myofibroblasts (E), or NHDF-Ad fibroblasts (F). The Ca²⁺ response was larger and ATZ-sensitive in HCT116 cancer cells. Data are the means of >50 cells. Error bars represent S.E.

Kinase Activity

Many kinase-operated signaling pathways are modulated by pH (65) and are expected to respond to changes in pCO₂. This was tested in HCT116 cells by measuring the phosphorylation state of the major mitogen-activated kinases, extracellular signal-regulated kinases, c-Jun N-terminal kinases, and p38 isoforms. pCO₂ was held constant at 5 or 20% or oscillated between 5 and 20% CO₂ for six cycles (4-min periodicity) in the presence or absence of ATZ to manipulate the dynamics of the pH_i response (note that at the end of pCO₂ oscillations cells were returned to 5% CO₂ for 2 min). After 26 min of superfusion, lysates were tested for kinase activity readouts to identify pH-sensitive and pH-insensitive kinases over the pH range studied. Among the kinases investigated, phosphorylation of ERK1 (Thr-202/Tyr-204) and ERK2 (Thr-185/Tyr-187) was unaffected by pCO₂ changes (Fig. 5A). In contrast, kinases, such as JNK1 and JNK2, were dephosphorylated (Thr-183/Tyr-185) by stably raised pCO₂. Transient exposure to 20% CO₂ (i.e. oscillating pCO₂) was inadequate to reduce phosphorylation, indicating that a sustained fall in pH_i is necessary for eliciting a change in JNK1/2 activity (Fig. 5B). Stably decreased pH_i also reduced the activity of mTOR complex 1 (mTORC1), reported as S6 kinase (S6K) phosphorylation at Thr-389 (65–67). Unlike the other kinases tested, mTORC1 signaling was dependent on CA_i activity under fluctuating pCO₂ protocols (higher S6K phosphorylation in the presence of ATZ; Fig. 5C; note that expression of the mTOR regulator GβL did not underlie differences in S6K phosphorylation). For the four pCO₂ protocols, S6K phosphorylation did not correlate with the time-averaged [H⁺]_i (Fig. 5C, panel ii), arguing that the dynamics of pH_i must be influencing the mTORC1 pathway. A single, transient (2-min) exposure to 20% CO₂ was sufficient for observing an effect of CA_i activity on mTORC1 (higher S6K phosphorylation in the presence of ATZ; Fig. 5D). The mTOR inhibitor rapamycin (10 μm) produced a substantial decrease in S6K phosphorylation (Fig. 5E), confirming the validity of using S6K as an mTORC1 readout. At constant (5%) pCO₂, ATZ did not affect mTORC1 activity (Fig. 5E), indicating that the ATZ sensitivity of S6K phosphorylation measured under oscillating pCO₂ is not an off-target effect of the CA inhibitor. Consistent with this finding, ATZ had no effect on S6K phosphorylation in CAII knockdown HCT116 (Fig. 5F) and wild-type MDA-MB-468 (Fig. 5G), i.e. cells with low CA_i activity. In summary, mTORC1 signaling responds to sharp pCO₂ changes that are attainable with high CA_i activity. These proof-of-principle experiments demonstrate that CA_i activity is able to modulate the coupling between extracellular pCO₂ (a function of blood flow) and intracellular signaling.

FIGURE 5.

Effects of CA_i-mediated CO₂-pH coupling on kinase phosphorylation. A, Phosphorylation state of ERK1 (Thr-202/Tyr-204) and ERK2 (Thr-185/Tyr-187) was unaffected by changing pCO₂ either tonically (5% or 20% for 30 min) or dynamically (six cycles of 5–20% fluctuations; cycle length of 4 min); densitometric analysis (n = 3). Time-averaged intracellular [H⁺] was measured in separate experiments. B, JNK1/2 were dephosphorylated (Thr-183/Tyr-185) by stably raised pCO₂ but unaffected by oscillating pCO₂ (n = 3). C, panel i, Western blot for S6 kinase phosphorylation (P) (readout of mTORC1 signaling) under 5% CO₂, 20% CO₂, and fluctuating pCO₂ (six cycles; 5–20%) in the presence or absence of ATZ (100 μm). Panel ii, pCO₂ fluctuations reduced S6 kinase phosphorylation but only under full CA_i activity. S6K activity did not correlate with time-averaged [H⁺] (n = 3). D, panels i and ii, analysis for one cycle of pCO₂ oscillation in HCT116 cells. E, at 5% CO₂, mTOR activity was unaffected by ATZ but strongly inhibited by rapamycin (10 μm). F, six cycles of pCO₂ fluctuations in CAII knockdown (KD) HCT116 cells (construct 695). G, six cycles of pCO₂ fluctuations in MDA-MB-468 cells (low CA_i activity). Error bars represent S.E. *, p > 0.05.

DISCUSSION

CA_i Activity in Cancer Cells

In this study, we quantified CA_i activity in a panel of cancer cells and compared these data with results from fibroblast and fibroblast-related cells. Activity measurements were performed under physiological conditions, in the native and undiluted cytoplasmic environment of intact cells, and in the presence of relevant regulatory cytoplasmic influences (Fig. 1A). CA_i activity varied considerably among cancer cell lines from essentially absent in HeLa and MDA-MB-468 cells to high in HCT116, HT29, and RT112 cells. Low CA_i activity was characteristic of fibroblasts/fibroblast-related cells, arguing that CA_i catalysis in solid tumors is more likely to be associated with cancer cells rather than stromal fibroblasts. Soluble (but not secreted) and cytoplasm-facing membrane-tethered CA isoforms may contribute toward the CA_i activity. Cancer cells with high total CA_i activity were also positive for soluble CA activity in membrane-free cell lysates (Fig. 1B) and for CAII immunoreactivity (Fig. 1D). Genetic knockdown of CAII in HCT116 cells demonstrated that the majority of CA_i activity was attributable to CAII (Fig. 1C). Additional isoforms may contribute to CA_i activity in other cell lines, such as HT1080 cells that lack CAII expression and soluble CA activity, but have measurable overall CA_i. Based on data from all cells investigated, CAII expression correlated strongly with soluble CA activity and was a good predictor of high total CA_i activity.

CA_i Activity Is Not Universally Rate-limiting for pH_i Regulation

The physiological role for CA_i activity in cancer is contentious although clearly distinct from that of exofacial CAs (6). Here, we explored the cellular processes that may depend on CA_i activity. Previous studies have argued for a role of CA_i activity in facilitating H⁺ or HCO₃⁻ transport across membranes (23–26) and H⁺ diffusion in cytoplasm (58, 59), but these interactions have not been tested robustly in cancer cells. CA_i activity in the six cancer cell lines studied does not correlate with resting pH_i, NHE flux, or HCO₃⁻ transporter flux measured previously in these cells (3). Our present data (Fig. 2) show that the H⁺ and HCO₃⁻ fluxes produced by cancer cells over the physiological pH_i range are not of sufficient magnitude to require CA_i catalysis, with the exception of acid loading by HCO₃⁻ export at high pH_i. In the absence of CA_i activity, the maximal capacity of CO₂ hydration to generate HCO₃⁻ ions for extrusion is ∼14 mm/min at 5% CO₂ (i.e. k_f × [CO₂]), and this can be rate-limiting for fast HCO₃⁻-dependent acid-loading transporters as measured in RT112 cells (Fig. 2A, panel iii). However, the highly alkaline intracellular conditions that are required for producing this CA_i dependence are unlikely to be typical of cancer cells. CA_i-catalyzed hydration of CO₂ has recently been proposed to facilitate Na⁺-HCO₃⁻ co-transport in cardiac myocytes by supplying the transport protein with H⁺ ions for titrating HCO₃⁻ (26). However, genetic CAII knockdown (HCT116; Fig. 2C, panel iv) or pharmacological inhibition with ATZ (RT112; Fig. 2A, panel iii) did not affect HCO₃⁻-dependent acid extrusion. This difference may reflect contrasting structural and chemical properties of cardiac and cancer cytoplasm. Alternatively, CA_i dependence may only be observed experimentally under high Na⁺-HCO₃⁻ co-transport fluxes, which are attainable in cardiac myocytes (under hyperkalemic stimulation) but not in cancer cells. NHE activity was able to produce rapid H⁺ extrusion in MDA-MB-468 cells that naturally have very low CA_i activity. This argues that CA_i-independent delivery of H⁺ ions does not limit the membrane transport process (Fig. 2B) in agreement with an earlier study on ventricular myocytes (26). In HCT116 cells (high CA_i activity), intrinsic cytoplasmic buffers alone supported the same magnitude of NHE flux as measured in the presence of CO₂/HCO₃⁻ or after loading cells with the highly mobile buffer carnosine (Fig. 2C). Even in the absence of CO₂/HCO₃⁻ (hence no CA_i catalysis), activation of NHE did not evoke measurable pH_i non-uniformity. This supports the case that diffusive H⁺ coupling across cancer cytoplasm is normally adequate with intrinsic (non-carbonic) buffers. Knockdown of CAII did not affect NHE activity in HCT116 cells (measured as the DMA-inhibitable component of flux; Fig. 2C, panel iv), arguing that neither catalysis nor the presence of CAII protein is necessary for high NHE fluxes. Collectively, these observations argue for the absence of rate-limiting barriers to H⁺ diffusion over the relatively small dimensions of cancer cells (mean radii, <10 μm). Additionally, cancer cells typically down-regulate gap junctions (22), which in other tissues allow for greatly expanded diffusion lengths. The absence of electrical coupling explains why CA_i catalysis cannot facilitate CO₂ or H⁺ diffusion appreciably in tumors as this would require HCO₃⁻ ions to diffuse relatively freely between cells.

CA_i Activity Influences the Degree of Coupling between pCO₂ and pH_i Dynamics

Most investigations of the role of CA_i activity in pH_i regulation have been performed under constant pCO₂. This condition may be appropriate for well perfused tissues with stable CO₂ production and venting but is not representative of metabolically active solid tumors with intermittent blood flow. Fluctuations in tumor blood flow are the basis for time-dependent changes in pO₂ (also known as acute hypoxia) that influence tumor biology (34). During periods of inadequate capillary washout, pCO₂ rises as O₂ is depleted, and these changes reverse upon blood reperfusion. Although direct, high resolution measurements of pCO₂ fluctuations are not available because of technical limitations, the inverse correlation between time-averaged pO₂ and pCO₂ (acidity) is well established (43–45). Our standard experimental protocol of varying pCO₂ between 5 and 20% with 4-min periodicity is based on measurements of extracellular pH, blood flow, and pO₂ in solid tumors in vivo. The pH of 20% CO₂ superfusate is 6.8, which is considered to be typical of the mean extracellular pH of most solid tumors (63, 68); pH 7.4 of 5% CO₂ superfusates is normal for blood plasma. Our choice of periodicity is in the range of fluctuation frequencies of blood flow and/or pO₂ established by Fourier analysis in tumors in vivo (30, 33, 42).

Changes in pCO₂ will have knock-on effects on [H⁺]_i dynamics and hence the many pH-sensitive downstream processes. The coupling between pCO₂ and pH_i is strongly dependent on CA_i as illustrated in Fig. 3 where the same experimental protocol of changing pCO₂ between 5 and 20% produced different [H⁺]_i responses depending on CA_i activity. [H⁺]_i fluctuations were slow and ATZ-insensitive in cells with very low CA_i activity (MDA-MB-468 and HeLa). In contrast, high CA_i activity in HCT116 and HT29 cells resulted in accentuated [H⁺]_i fluctuations that were dampened in the presence of ATZ. The effect of ATZ was not due to inhibition of exofacial CAs because (i) superfusates were pre-equilibrated (obviating the need for CA_e catalysis) and (ii) [H⁺]_i dynamics were ATZ-insensitive in CAIX-expressing MDA-MB-468 cells.

Mathematical modeling demonstrates that the effect of CA_i activity on [H⁺]_i dynamics was important even with a smoother sinusoidal pCO₂ waveform (Fig. 3G), which is more representative of undulating blood flow. The ability of CA_i to influence [H⁺]_i dynamics was predicted for a wide range of pCO₂ waveform amplitudes but required a periodicity of 4 min or less for physiologically meaningful effects (Fig. 3H). This frequency dependence is expected because even at spontaneous reaction rates, [H⁺]_i is able to track slow changes in pCO₂. Given that fluctuations of blood flow (29–32) and pO₂ (30, 32, 34–41) in solid tumors have been observed with a periodicity as short a 0.5 min (i.e. two cycles/min), a range of effects on [H⁺]_i dynamics could be achieved by regulating CA_i activity: from tight temporal pCO₂-pH_i coupling at high CA_i activity to low pass filtering with negligible CA_i activity. Cancer cells would be able to tune their pH_i responsiveness to changes in pCO₂ (blood flow) by regulating CA_i activity, e.g. through CAII expression.

Our study explored the functional significance of the CA_i dependence of [H⁺]_i dynamics using Ca²⁺ signaling and kinase-operated cascades as proof-of-principle examples (Fig. 6). In HCT116 cells, a transient pH_i rise, evoked by reducing pCO₂, increased [Ca²⁺]_i. Our evidence points to store-operated calcium entry as the mechanism triggering the [Ca²⁺]_i response (Fig. 4C). Recent work has demonstrated that store-operated calcium entry is reduced at low pH_i because of an uncoupling between the endoplasmic reticulum Ca²⁺ sensor STIM and the surface membrane Ca²⁺ channel Orai (64). Because [Ca²⁺]_i responds dynamically to changes in the balance between influx and efflux pathways, pH_i sensitivity of Ca²⁺ entry would produce the observed [Ca²⁺]_i fluctuations. Under CA_i inhibition with ATZ, the same pCO₂ stimulus evoked a slower and smaller [Ca²⁺]_i response.

FIGURE 6.

Effect of CA_i activity on translating dynamic changes in pCO₂ (e.g. due to intermittent blood flow) into pH_i dynamics and thenceforth on signaling cascades (e.g. Ca²⁺ and mTOR signaling).

Previous studies have shown that the environmental sensor mTOR, which is strongly linked with cancer (66, 69, 70), is inhibited at low pH_i (65). The present work shows that the degree of mTOR inhibition also depends on CA_i activity when pCO₂ is fluctuating. In HCT116 cells, a single cycle of pCO₂ oscillation produced a lower readout of mTORC1 signaling (S6 kinase phosphorylation) when CA_i activity was intact (Fig. 5D). This inhibitory effect of CA_i catalysis on mTORC1 signaling was substantiated by expanding the protocol to six cycles of pCO₂ fluctuations (Fig. 5C). mTORC1 signaling was generally higher when pCO₂ oscillations were performed on HCT116 cells with pharmacologically (ATZ) or genetically (knockdown; Fig. 5F) reduced CA_i activity or on cells with naturally low CA_i activity, such as MDA-MB-468 (Fig. 5G). mTORC1 activity was not a unique function of time-averaged pH_i. For example, holding pCO₂ stably at 20% or fluctuating pCO₂ between 5 and 20% in the presence of ATZ produced a similar time-averaged pH_i, but S6 kinase phosphorylation differed by a factor of 2 (Fig. 5C, panel ii). We conclude that the dynamics of pH_i changes must influence mTORC1. Akin to the notion that transient hypoxia (perfusion-limited) and stable hypoxia (diffusion-limited) have distinct biological consequences (71), there may be similar “frequency modulation” and “amplitude modulation” (72) aspects of H⁺ signaling in cancer. A possible explanation why some kinases (such as JNK1/2) do not respond to fluctuating pCO₂ is a slow binding of H⁺ ions to modulatory sites that acts like a low pass filter. mTOR may be able to register rapid pH_i fluctuations by faster H⁺ binding kinetics. Further studies are necessary to explore these possible mechanisms.

CA_i-catalyzed CO₂ hydration will produce fluctuations in [HCO₃⁻] that parallel pH_i changes, and it is plausible that HCO₃⁻ ions interact with targets in a pH-independent manner. Removing HCO₃⁻ ions from cytoplasm also removes the substrate (CO₂) for CA_i; therefore a simple buffer substitution experiment would not be an appropriate test to distinguish the effects of H⁺ and HCO₃⁻ ions. However, it is generally accepted that H⁺ ions are more reactive than HCO₃⁻ ions. Furthermore, earlier observations of the acid response of store-operated calcium entry (64) and mTORC1 (65) were made in the absence of CO₂/HCO₃⁻, arguing for H⁺ ions as the key regulators.

Growing evidence points to a negative correlation between the expression of CA_i isoforms, such as CAII, and cancer disease severity (11, 13–15). Importantly for disease progression, CAII down-regulation would help to protect cytoplasmic pH from pCO₂ fluctuations and bestow cancer cell pH with a greater degree of autonomy from extracellular influences, such as those arising from intermittent blood flow (73). Conversely, rapid changes in blood flow would be registered by cells with high CA_i activity. For example, mTOR-regulated metabolism and proliferation would be more responsive to fluctuating blood flow in cancer cells with higher CA_i activity, particularly in regions close to aberrant blood vessels, which experience the sharpest pCO₂ fluctuations. Incidentally, these tumor regions are also important for metastasis and nutrient sensing. In solid tumors made of cancer cells with high CA_i activity surrounded by a fibroblast stroma of low CA_i activity, pCO₂ fluctuations would evoke distinct [H⁺] responses in the two cell types as illustrated by [Ca²⁺]_i responses (Fig. 4).

In conclusion, this study demonstrates a novel role for CA_i activity in cancer as a transducer of pCO₂ fluctuations (which arise from intermittent blood flow) into a potent cytoplasmic signal (H⁺ ions). Cancer cells may alter the degree of temporal coupling between pCO₂ and pH_i by tuning cytoplasmic CA_i activity, e.g. through CAII expression (Fig. 6). This work highlights the importance of dynamic aspects of pH signaling as a modality distinct from responses to stable pH changes.