Fall In pHi Research Articles

Modulation of acid extrusion and acid loading in neurons and astrocytes by RPTPγ and RPTPζ

Receptor protein tyrosine phosphatases γ and ζ (RPTPγ & RPTPζ) are transmembrane signaling proteins that, as monomers, catalyze dephosphorylation of tyrosine residues on cytosolic targets. RPTPγ and RPTPζ are unusual in having an extracellular carbonic-anhydrase–like domain (CALD). Carbonic anhydrases (CAs) catalyze the reaction, CO₂ + H₂O ⇌ HCO3– + H⁺, but CALDs are enzymatically inactive. Previous studies on renal proximal tubules (PTs) strongly suggest that RPTPγ is a dual extracellular CO₂/HCO₃⁻ sensor. RPTPγ—expressed on the PT basal (i.e., blood-side) membrane—is essential for modulating acid-base transport in response to changes in basolateral [CO₂] and [HCO₃⁻]. RPTPγ and RPTPζ are also present in the central nervous system (CNS), including mouse hippocampus (HC). RPTPγ is expressed primarily in neurons and RPTPζ, in astrocytes. Here, we investigate whether RPTPγ and RPTPζ in brain, like RPTPγ in kidney, play roles in sensing extracellular [CO₂]ₒ and [HCO₃⁻]ₒ and altering acid-base transport. We hypothesize that activation/termination of the intracellular phosphatase activity of these RPTPs regulates initial steps in signaling cascades that defend cells against intracellular pH (pHi) decreases triggered by metabolic acidosis (MAc, caused by ↓ [HCO₃⁻]ₒ) or respiratory acidosis (RAc, caused by ↑ [CO₂]ₒ). We propose that the RPTPs respond to ↑[CO₂]ₒ or ↓[HCO₃⁻]ₒ by promoting a shift from dimers to monomers, thereby dis-inhibiting the activity of the intracellular phosphatase domains. The ultimate result of the RPTP dis-inhibition would be increased acid-extrusion rate (JE) or decreasedacid-loading rate (JL), both of which would minimize the fall of pHi during a first exposure to MAc or RAc, and may promote adaptation during a second exposure. Immunocytochemistry of mixed HC neuron-astrocyte cultures from wild-type (WT) mice using a primary antibody against the RPTPγ fibronectin III domain shows that RPTPγ colocalizes with MAP2 in HC neurons but not with GFAP in HC astrocytes . RT-PCR cloning from mixed HC cultures identifies specific RPTPγ and RPTPζ variants. Presumably it is the neurons that express two identified RPTPγ variants: the full-length protein, encoded by all 30 ptprg exons (NM_008981), and a second newly-detected variant that corresponds to the hypothetical assembly X1 (XM_006517956). The HC co-cultures also express six RPTPζ variants: Three RPTPζ variants (V) have previously been validated in mice: VR3 (NM_001081306), VR4 (NM_001311064), and VR5 (NM_001361349). Three others were previously only reported as hypothetical assemblies: X2 (XM_006505013), X3 (XM_006505014), and X4 (XM_006505015). To assess the influence of RPTPζ on pHi homeostasis, we loaded cells with the pH-sensitive dye BCECF, digitally monitored dye fluorescence, and computed pHi of cells subjected to acid-base disturbances. In MAc-MAc protocols, the effects of the knockout (KO) of RPTPζ are unremarkable in both neurons and astrocytes. In RAc-RAc protocols, the KO has unremarkable effects on neurons. However, in astrocytes the RPTPζ-KO markedly increases both the rates and the magnitudes of pHi decrease during both RAc pulses. Thus, RPTPζ may stabilize pHi in astrocytes subjected to RAc.

Dynamic Regulation of Intracellular PH in the Heart

Intracellular pH (pHi) is a key regulator of cellular functions. Cellular proteins rely on pHi to maintain their structures and functions. The mammalian heart beats incessantly with rhythmic mechanical activities generating acids that need to be buffered and removed from the cytosol to maintain a relatively stable pHi for normal cardiac function. Cardiac pHi is critical to cardiac excitation and contraction, and an uncompensated fall in pHi in cardiac ischemia impairs cardiac function and trigger arrhythmia and sudden cardiac death. It was reported that there exists spatial pHi non-uniformity in cardiomyocytes. However, it remains unknown how cardiac cells handle pHi on a beat-to-beat basis. We designed a method to measure simultaneous sarcomere contraction and pHi in cardiomyocytes. We found dynamic beat-to-beat acidification of pHi during a cardiac cycle, namely “pHi transients”. The pHi transients were coupled with cardiac contraction and regulated by pHi buffering capacity, pacing rate, HCO3-/Cl- transporters, and β-adrenergic signaling. Inhibition of the mitochondrial electron-transport chain significantly attenuates the pHi transients. Indeed, the dynamic changes in pHi during cardiac contraction reflects the rhythmic metabolic status in cardiomyocytes. Physiologically, the temporal pHi acidification may provide a beat-to-beat increase in the driving force for H+ transport to enhance mitochondrial ATP synthesis during cardiac contraction. Moreover, the beat-to-beat transient intracellular acidification may provide a negative feedback mechanism to cardiac contraction by facilitating relaxation due to the inhibitory effects on the contractile machinery by low pHi. Thus, in addition to dynamic electrical (action potentials), Ca2+ (Ca2+ transients), and mechanical regulatory systems (contractions), our study identified and demonstrated a new dynamic regulatory system (“pHi transients”) in the heart. Our results show that a cardiac cycle is sculpted not only by action potentials, Ca2+ transients, and contractions but also by cyclical changes in pHi.

Cancer and Alzheimer's disease: intracellular pH scales the metabolic disorders.

Alzheimer's disease (AD) and cancer have much in common than previously recognized. These pathologies share common risk factors (inflammation and aging), with similar epidemiological and biochemical features such as impaired mitochondria. Metabolic reprogramming occurs during aging and inflammation. We assume that inflammation is directly responsible of the Warburg effect in cancer cells, with a decreased oxidative phosphorylation and a compensatory highthroughput glycolysis (HTG). Similarly, the Warburg effect in cancer is thought to support an alkaline intracellular pH (pHi), a key component of unrelenting cell growth. In the brain, inflammation results in increased secretion of lactate by astrocytes. The increased uptake of lactic acid by neurons results in the inverse Warburg effect, such as seen in AD. The neuronal activity is dampened by a fall of pHi. Pronounced cytosol acidification results in decreased mitochondrial energy yield as well as apoptotic cell death. The link between AD and cancer is reinforced by the fact that treatment aiming at restoring the mitochondrial activity have been experimentally shown to be effective in both diseases. Low carb diet, lipoic acid, and/or methylene blue could then appear promising in both sets of these clinically diverse diseases.

Investigation of the Intracellular‐pH (pHi) Dependence of the Electrogenic Sodium Bicarbonate Cotransporter NBCe1‐A

Maintaining intracellular pH (pHi) within a narrow range is essential for many cellular processes to occur. The fine‐tuning of pHi is the result of the simultaneous action of a variety of cellular pH buffers, that tend to minimize changes in pHi, and transporters that adjust their rates to move acid‐base equivalents across the plasma membrane, tending to stabilize pHi. The electrogenic Na/HCO3 cotransporter (NBCe1), by moving HCO3− into or out of a cell, is a major player in pHi regulation. In addition, NBCe1 often plays a central role in transepithelial HCO3− transport. The purpose of the present study is to investigate the pHi‐dependence of NBCe1‐A, the variant of NBCe1 mainly expressed in the renal proximal tubule. We inject Xenopus oocytes with H2O (control) or cRNA encoding NBCe1‐A. A few days later, we use ion‐selective microelectrodes and two‐electrode‐voltage clamping to simultaneously monitor pHi and the ionic current carried by NBCe1‐A (INBC). We take advantage of out‐of‐equilibrium (OOE) CO2/HCO3− solutions to keep extracellular [Na+] ([Na+]o), pHo, and [HCO3−]o fixed as we change [CO2]o from (a) 0% (“pure” HCO3−) to (b) 2.5% (‘OOE1’), or (c) 5% (standard equilibrated solution, ‘EQ’), or (d) 10% (‘OOE2’), or (e) 20% (‘OOE3’). The use of OOE solutions containing different [CO2]o allows us to manipulate the nadir value that pHi reaches after CO2 equilibration (i.e., the higher the [CO2]o, the lower the pHi after CO2 equilibration). Our experimental protocol is the following. After obtaining a first set of voltage‐clamp recordings (IV#1) in the continuous presence of ND96 (CO2/HCO3− free), we switch the superfusion solution from ND96 to “pure” HCO3− (0% CO2/33 mM HCO3−/pH 7.50). For an oocyte expressing NBCe1‐A, the entry of HCO3− into the cell via NBCe1‐A causes pHi to rise and the membrane potential (Vm) to become more negative. As pHi reaches ~7.50, we obtain a second set of voltage‐clamp recordings (IV#2). Following IV#2, we switch the superfusion solution from “pure” HCO3− to a CO2‐containing solution (‘OOE1’, ‘EQ’, ‘OOE2’ or ‘OOE3’). After ~5 min, once pHi reaches its nadir and before the typical pHi recovery caused by HCO3− entry into the cell, we obtain a third set of voltage‐clamp recordings (IV#3). For a H2O‐injected oocyte, which has no appreciable pathways for HCO3− movements through the plasma membrane, exposure to “pure” HCO3− causes only a very slow and tiny fall in pHi, due to a tiny amount of contaminating CO2 in the “pure” HCO3− solution, and no hyperpolarization; exposure to a CO2‐containing solution causes pHi to decay. For each experiment, we calculate the HCO3−‐dependent conductance (G) as an index of NBCe1‐A activity. We account for different NBCe1‐A expression levels, by normalizing G in CO2‐containing solution (G from IV#3) to G in “pure” HCO3− (G from IV#2). Analysis of normalized G versus pHi shows no correlation between these two quantities. Thus, we reach the surprising conclusion that NBCe1‐A is not pHi sensitive in the pH range of our experiments (i.e., 6.70–7.50).Support or Funding InformationSupported by NIH K01‐DK107787 to RO and NIH R01‐DK113197, ONR N00014‐15‐1‐2060, ONR N00014‐16‐1‐2535 to WFBThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Na‐driven Cl/HCO3 exchanger (NDCBE) activity is essential for establishing new steady‐state in intracellular pH during metabolic acidosis in murine hippocampal neurons and astrocytes

The Na‐driven Cl/HCO3 exchanger (NDCBE) is highly expressed in CA1 hippocampal (HC) neurons but has not been reported to be present in astrocytes. NDCBE extrudes acid from the cytoplasm by mediating the exchange of 1 Na+ and 2 HCO3− (or 1 CO3=) from the extracellular compartment for 1 Cl− from the intracellular compartment. Previously, we showed that knocking‐out anion exchanger 3 (AE3), which is an acid loader expressed only in neurons but not astrocytes, reduces the ability of HC neurons and astrocytes to acid load the cytoplasm. We also showed that, after 70 sec in metabolic acidosis (MAc), intracellular pH (pHi) in neurons and astrocytes from AE3−/− mice decreases continuously over 7 min (i.e., fails to stabilize). We hypothesized that this failure to reach new steady‐state is due to a decreased ability of neurons and astrocytes to increase their acid‐extrusion rate (JE) sufficiently to balance the acid‐loading rate (JL). In the present study we investigate the role of NDCBE activity in facilitating sufficient JE to balance JL during MAc. We induced MAc by decreasing the extracellular [HCO3−] from 22 mM to 14 mM at fixed 5% CO2. This maneuver decreases extracellular pH from 7.4 to 7.2. We monitored the changes in pHi during MAc exposure using fluorescence imaging of the pH sensitive dye BCECF. We obtained co‐culture of neurons and astrocytes from the hippocampal CA1 regions from wild‐type (WT) and NDCBE‐knockout (NDCBE−/−) mice. Our results show that, for neurons, the initial steady‐state pHi is significantly higher in WT (7.25 ± 0.03) compared to NDCBE−/− (7.01 ± 0.01). However, the MAc‐induced acidification rate during the first 70 sec of the MAc (dpHi/dt)early is not significantly different between WT (−1.6 × 10−3 ± 0.8× 10−3 pH/s) and NDCBE−/− (−1.4 × 10−3 ± 0.9 × 10−3 pH/s). After 70 sec in MAc, the pHi of NDCBE−/− decreases continuously over 7 min, with a slope, (dpHi/dt)late, of −3.0 × 10−4 ± 0.6 × 10−4 pH/s, which is significantly faster than WT neurons (−0.9 × 10−4 ± 0.6 × 10−4 pH/s). For astrocytes, the knockout of NDCBE surprisingly reduces initial steady‐state from 6.99 ± 0.01 for the WT to 6.90 ± 0.01 for NDCBE−/− and significantly reduces (dpHi/dt)early from −3.3 × 10−3 ± 0.2 × 10−3 pH/s in the WT to −2.2 × 10−3 ± 0.1 × 10−3 in NDCBE−/−. The results also reveal that the absence of NDCBE reduces the astrocytes ability to stabilize pHi after 70 sec in MAc, with a (dpHi/dt)late of −2.0 × 10−4 ± 0.3 × 10−4 pH/s compared to the WT (dpHi/dt)late of −0.6× 10−4 ± 0.6 × 10−4 pH/s. Our results are consistent with the hypothesis that NDCBE activity is essential for braking the fall of pHi in both HC neurons and astrocytes during MAc exposure.Support or Funding InformationR01‐DK081567, R01‐NS018400 and T32 HL007913

NH3 Permeability versus CO2 Permeability: Insights from Mathematical Modeling

Exposing a cell to a solution containing NH3/NH4+ leads to a rapid NH3 influx, which causes a rise in intracellular pH (pHi) and a transient decrease in surface pH (pHS). Conversely, exposing a cell to a solution containing CO2/HCO3− leads to a rapid CO2 influx, which causes a fall in pHi and a transient increase in pHS.In our laboratory we use the maximal change in the height of the pHS transient (ΔpHS) as a semiquantitative index of membrane permeability to NH3 (PM,NH3) or to CO2 (PM,CO2). As a first step in extracting absolute membrane permeability from pHS measurements, in 2012 we developed a reaction‐diffusion mathematical model of CO2 influx to investigate the dependence of ΔpHS predicted by the model on PM,CO2 values (Somersalo et al, J Theor Biol, 2012). To account for physiological data on gas channels, the model predicts that,contrary to widespread assumptions, the membrane must offer considerable resistance to the movement of CO2.Here, we investigate the dependence of ΔpHS versus PM,NH3 with a reaction‐diffusion mathematical model of NH3 influx into a spherical cell. The model is an extension of the model of Somersalo et al, in which now we include the NH3/NH4+ buffer equilibrium and passive transmembrane flux of NH3. After having checked that the model correctly predicts the transient changes in pHi and pHS that one would expect as NH3 enters a cell, we performed simulations in which we systematically decreased PM,NH3 from that corresponding to a thin film of water. By comparing the dependence of ΔpHS versus a range of PM,NH3 (present study) with the dependence of ΔpHS versus PM,CO2 (Somersalo et al), we conclude that, contrary to the Overton's prediction (which considers only solubility in the membrane), the background PM,NH3 (in absence of gas channels) is far higher than the background PM,CO2.

Evidence from simultaneous intracellular- and surface-pH transients that carbonic anhydrase II enhances CO2 fluxes across Xenopus oocyte plasma membranes.

The α-carbonic anhydrases (CAs) are zinc-containing enzymes that catalyze the interconversion of CO2 and HCO3 (-). Here, we focus on human CA II (CA II), a ubiquitous cytoplasmic enzyme. In the second paper in this series, we examine CA IV at the extracellular surface. After microinjecting recombinant CA II in a Tris solution (or just Tris) into oocytes, we expose oocytes to 1.5% CO2/10 mM HCO3 (-)/pH 7.50 while using microelectrodes to monitor intracellular pH (pHi) and surface pH (pHS). CO2 influx causes the familiar sustained pHi fall as well as a transient pHS rise; CO2 efflux does the opposite. Both during CO2 addition and removal, CA II increases the magnitudes of the maximal rate of pHi change, (dpHi/dt)max, and the maximal change in pHS, ΔpHS. Preincubating oocytes with the inhibitor ethoxzolamide eliminates the effects of CA II. Compared with pHS, pHi begins to change only after a delay of ~9 s and its relaxation has a larger (i.e., slower) time constant (τpHi > τpHS ). Simultaneous measurements with two pHi electrodes, one superficial and one deep, suggest that impalement depth contributes to pHi delay and higher τpHi . Using higher CO2/HCO3 (-) levels, i.e., 5%/33 mM HCO3 (-) or 10%/66 mM HCO3 (-), increases (dpHi/dt)max and ΔpHS, though not in proportion to the increase in [CO2]. A reaction-diffusion mathematical model (described in the third paper in this series) accounts for the above general features and supports the conclusion that cytosolic CA-consuming entering CO2 or replenishing exiting CO2-increases CO2 fluxes across the cell membrane.

Evidence from simultaneous intracellular- and surface-pH transients that carbonic anhydrase IV enhances CO2 fluxes across Xenopus oocyte plasma membranes.

Human carbonic anhydrase IV (CA IV) is GPI-anchored to the outer membrane surface, catalyzing CO2/HCO3 (-) hydration-dehydration. We examined effects of heterologously expressed CA IV on intracellular-pH (pHi) and surface-pH (pHS) transients caused by exposing oocytes to CO2/HCO3 (-)/pH 7.50. CO2 influx causes a sustained pHi fall and a transient pHS rise; CO2 efflux does the opposite. Both during CO2 addition and removal, CA IV increases magnitudes of maximal rate of pHi change (dpHi/dt)max, and maximal pHS change (ΔpHS) and decreases time constants for pHi changes (τpHi ) and pHS relaxations (τpHS ). Decreases in time constants indicate that CA IV enhances CO2 fluxes. Extracellular acetazolamide blocks all CA IV effects, but not those of injected CA II. Injected acetazolamide partially reduces CA IV effects. Thus, extracellular CA is required for, and the equivalent of cytosol-accessible CA augments, the effects of CA IV. Increasing the concentration of the extracellular non-CO2/HCO3 (-) buffer (i.e., HEPES), in the presence of extracellular CA or at high [CO2], accelerates CO2 influx. Simultaneous measurements with two pHS electrodes, one on the oocyte meridian perpendicular to the axis of flow and one downstream from the direction of extracellular-solution flow, reveal that the downstream electrode has a larger (i.e., slower) τpHS , indicating [CO2] asymmetry over the oocyte surface. A reaction-diffusion mathematical model (third paper in series) accounts for the above general features, and supports the conclusion that extracellular CA, which replenishes entering CO2 or consumes exiting CO2 at the extracellular surface, enhances the gradient driving CO2 influx across the cell membrane.

Ca2+-pumping impairment during repetitive fatiguing contractions in single myofibers: role of cross-bridge cycling

The energy cost of contractions in skeletal muscle involves activation of both actomyosin and sarcoplasmic reticulum (SR) Ca²⁺-pump (SERCA) ATPases, which together determine the overall ATP demand. During repetitive contractions leading to fatigue, the relaxation rate and Ca²⁺ pumping become slowed, possibly because of intracellular metabolite accumulation. The role of the energy cost of cross-bridge cycling during contractile activity on Ca²⁺-pumping properties has not been investigated. Therefore, we inhibited cross-bridge cycling by incubating isolated Xenopus single fibers with N-benzyl-p-toluene sulfonamide (BTS) to study the mechanisms by which SR Ca²⁺ pumping is impaired during fatiguing contractions. Fibers were stimulated in the absence (control) and presence of BTS and cytosolic calcium ([Ca²⁺]c) transients or intracellular pH (pHi) changes were measured. BTS treatment allowed normal [Ca²⁺]c transients during stimulation without cross-bridge activation. At the time point that tension was reduced to 50% in the control condition, the fall in the peak [Ca²⁺]c and the increase in basal [Ca²⁺]c did not occur with BTS incubation. The progressively slower Ca²⁺ pumping rate and the fall in pHi during repetitive contractions were reduced during BTS conditions. However, when mitochondrial ATP supply was blocked during contractions with BTS present (BTS + cyanide), there was no further slowing in SR Ca²⁺ pumping during contractions compared with the BTS-alone condition. Furthermore, the fall in pHi was significantly less during the BTS + cyanide condition than in the control conditions. These results demonstrate that factors related to the energetic cost of cross-bridge cycling, possibly the accumulation of metabolites, inhibit the Ca²⁺ pumping rate during fatiguing contractions.

Immunocytochemical techniques identify Na+‐coupled HCO3− transporters (NCBTs) in chemosensitive neurons of the Medullary Raphé

The medullary raphé of the medulla oblongata contains chemosensitive neurons that sense Δ[CO2]. Serotonergic (5HT) neurons increase their firing rate in response to increases in arterial [CO2]. The GABAergic response is a decreased firing rate. Both contribute to increased pulmonary ventilation. The neuronal electroneutral Na/HCO3− cotransporters (nNCBTs) respond to intracellular acid loads (e.g. caused by increased [CO2]) by actively moving HCO3− into cells to restore the intracellular pH. We hypothesized that nNCBTs are less abundant in serotonergic and GABAergic neurons than in non‐chemosensitive neurons. A reduced nNCBT presence would be expected to slow pHi recovery, sustaining the fall in pHi and maintaining altered firing rates in 5HT and GABA neurons. An immunocytochemistry protocol was developed to distinguish cell types using monoclonal primary antibodies against MAP2, a neuronal marker; TPH, a 5HT neuronal marker; and GAD2, a GABA neuronal marker. The cell type–specific staining was colocalized with polyclonal antibodies against the nNCBTs: NBCn1, NBCn2, and NDCBE. Quantitative image analysis techniques were applied to distinguish positively stained cells from the background signals. Contrary to the hypothesis, the fraction of nNCBT‐positive cells was >80% in all cell populations for all 3 nNCBTs, and not substantially different for TPH+ vs TPH− neurons or GAD2+ vs GAD2−.

A Reaction‐Diffusion Model of Acid‐Base Balance in a Xenopus Oocyte

We present a new three-dimensional mathematical model of a Xenopus oocyte. The model assumes that the oocyte is a perfectly symmetric spherical cell surrounded by the extracellular unconvected solution (EUS) which in turn is surrounded by the stirred bulk extracellular solution (BECS). The oocyte plasma membrane is assumed to be infinitely thin and permeable only to CO2. Using Fick's second law of diffusion and the law of mass action, diffusion and reaction processes are modeled in both EUS and intracellular fluid (ICF). The model accounts for the slow equilibration CO2 + H2O ⇄ H2CO3 as governed by the forward and reverse rate constants and for competing equilibria among the HCO3− and an indefinite number of non-HCO3− buffers. We present the results of numerical simulations when, in addition to the CO2/HCO3− buffer, a single mobile non-HCO3− buffer is present in the ICF and EUS. By solving the system of reaction-diffusion equations we analyze how the profiles of pH and solutes change in time and space as a result of the CO2 influx. Consistent with experimental data (Musa-Aziz et al, PNAS, 2009; ASN 2005); the model predicts that applying extracellular 1.5% CO2/10 mM HCO3− (fixed pH of 7.50) causes the familiar fall in pHi and a rise followed by decay in pHS. We also show how the presence of two non-HCO3− intracellular buffers with different mobilities and how the CO2 membrane permeability affect the predictions of the model.

Estimation of Acid‐Base and NH+4 Transport Parameters in a TAL Cell Model Using Inverse Methods

A mathematical model of a medullary thick ascending limb (mTAL) cell was used to study cytosolic pH (pHi) responses to luminal NH4+ perturbations. TAL cells load NH4+ via apical NH4+‐permeable channels and the NKCC2 cotransporter, resulting in a rapid fall in pHi. Our goal was to use our model and published NH4+ perturbation data to estimate parameters associated with pHi regulation using an inverse method. The TAL cell model, which includes NH+4 binding by NKCC2 and NHE, also includes models for Na+,K+‐ATPase, the Cl−‐HCO3− exchanger, KCC1, and trans‐ and paracellular electrodiffusion. A least‐squares problem was formulated and solved to fit the model to the data. First, steady‐state short‐circuit current data was used to estimate parameters important in Na+ transport; this yielded reasonable cytosolic concentrations, membrane PD's and resistances. The model was then fit to time records of NH4+ perturbations, with and without inhibition of NKCC2 or K+ channels, to estimate parameters associated with NH4+ transport. The results show that our TAL cell model, when used with parameter sets obtained from optimization methods, exhibits behaviors consistent with a wide variety of experimental results, including NH4+ perturbations, in isolated perfused TAL segments. This provides validation of the TAL model, which we intend to use in a multi‐cell model of the TAL segment.

Concentration-Dependent Effects on Intracellular and Surface pH of Exposing Xenopus oocytes to Solutions Containing NH3/NH4 +

Others have shown that exposing oocytes to high levels of NH(3)/NH(4)(+) (10-20 mM) causes a paradoxical fall in intracellular pH (pH(i)), whereas low levels (e.g., 0.5 mM) cause little pH(i) change. Here we monitored pH(i) and extracellular surface pH (pH(S)) while exposing oocytes to 5 or 0.5 mM NH(3)/NH(4)(+). We confirm that 5 mM NH(3)/NH(4)(+) causes a paradoxical pH(i) fall (-DeltapH(i) approximately equal 0.2), but also observe an abrupt pH(S) fall (-DeltapH(S) approximately equal 0.2)-indicative of NH(3) influx-followed by a slow decay. Reducing [NH(3)/NH(4)(+)] to 0.5 mM minimizes pH(i) changes but maintains pH(S) changes at a reduced magnitude. Expressing AmtB (bacterial Rh homologue) exaggerates -DeltapH(S) at both NH(3)/NH(4)(+) levels. During removal of 0.5 or 5 mM NH(3)/NH(4)(+), failure of pH(S) to markedly overshoot bulk extracellular pH implies little NH(3) efflux and, thus, little free cytosolic NH(3)/NH(4)(+). A new analysis of the effects of NH(3) vs. NH(4)(+) fluxes on pH(S) and pH(i) indicates that (a) NH(3) rather than NH(4)(+) fluxes dominate pH(i) and pH(S) changes and (b) oocytes dispose of most incoming NH(3). NMR studies of oocytes exposed to (15)N-labeled NH(3)/NH(4)(+) show no significant formation of glutamine but substantial NH(3)/NH(4)(+) accumulation in what is likely an acid intracellular compartment. In conclusion, parallel measurements of pH(i) and pH(S) demonstrate that NH(3) flows across the plasma membrane and provide new insights into how a protein molecule in the plasma membrane-AmtB-enhances the flux of a gas across a biological membrane.

Comparison of pH-dependence of Carbonic Anhydrase Activity in vitro and in Living Cells

Carbonic anhydrase (CA) plays an important role in acid-base homeostasis. It catalyses carbonic buffering, increases intracellular H+-mobility and facilitates the activity of pHi regulatory transporters. In vitro, CA activity is known to be inhibited by a fall of pH. This observation has been largely overlooked when considering mechanisms of pHi regulation. There is no information, however, on the pH-sensitivity of CA in intact cells. We have therefore investigated the pH-sensitivity of CA both in vitro, and in situ in living cells. In vitro CA activity was assessed from the time-course of pH change after addition of CO2-saturated water to a buffered solution (pH 8.0-6.5) containing purified bovine CA2, membrane fragments of HCT116 cells stably transfected with CA9, or cardiac homogenates from rat ventricle. In situ CA activity was assessed from the rate of CO2-induced fall of pHi in rat superfused ventricular myocytes, AM-loaded with the pH-fluorophore, carboxy-SNARF-1. The pH-sensitivity was estimated by fitting a kinetic algorithm to the pH time-course data. Results show that both in vitro and in situ CA-activity displays strong pH-dependence, activity declining with a fall of pH. Apparent pK and Hill coefficient (nH) values for in vitro pH-dependence were 7.00 and 1.10 respectively for purified CA2, 6.85 & 1.73 for CA9, and 7.15 and 0.80 for cardiac homogenates. The intracellular pKa and nH values for generic cardiac CA were 7.20 and 2.50. These results imply that the enzyme's contribution to both intracellular and extracellular pH-regulation may vary dynamically over the physiological pH range. Work funded by British Heart Foundation, Cancer Research UK, and Royal Society.

Effects of anoxia, aglycemia, and acidosis on cytosolic Mg2+, ATP, and pH in rat sensory neurons

Sensory neurons can detect ischemia and transmit pain from various organs. Whereas the primary stimulus in ischemia is assumed to be acidosis, little is known about how the inevitable metabolic challenge influences neuron function. In this study we have investigated the effects of anoxia, aglycemia, and acidosis upon intracellular Mg(2+) concentration [Mg(2+)](i) and intracellular pH (pH(i)) in isolated sensory neurons. Anoxia, anoxic aglycemia, and acidosis all caused a rise in [Mg(2+)](i) and a fall in pH(i). The rise in [Mg(2+)](i) in response to acidosis appears to be due to H(+) competing for intracellular Mg(2+) binding sites. The effects of anoxia and aglycemia were mimicked by metabolic inhibition and, in a dorsal root ganglia (DRG)-derived cell line, the rise in [Mg(2+)](i) during metabolic blockade was closely correlated with fall in intracellular ATP concentration ([ATP](i)). Increase in [Mg(2+)](i) during anoxia and aglycemia were therefore assumed to be due to MgATP hydrolysis. Even brief periods of anoxia (<3 min) resulted in rapid internal acidosis and a rise in [Mg(2+)](i) equivalent to a decline in MgATP levels of 15-20%. With more prolonged anoxia (20 min) MgATP depletion is estimated to be around 40%. With anoxic aglycemia, the [Mg(2+)](i) rise occurs in two phases: the first beginning almost immediately and the second after an 8- to 10-min delay. Within 20 min of anoxic aglycemia [Mg(2+)](i) was comparable to that observed following complete metabolic inhibition (dinitrophenol + 2-deoxyglucose, DNP + 2-DOG) indicating a near total loss of MgATP. The consequences of these events therefore need to be considered in the context of sensory neuron function in ischemia.

Cloning and characterization of an electrogenic Na/HCO3− cotransporter from the squid giant fiber lobe

The squid giant axon is a classic model system for understanding both excitable membranes and ion transport. To date, a Na(+)-driven Cl-HCO(3)(-) exchanger, sqNDCBE--related to the SLC4 superfamily and cloned from giant fiber lobe cDNA--is the only HCO(3)(-)-transporting protein cloned and characterized from a squid. The goal of our study was to clone and characterize another SLC4-like cDNA. We used degenerate PCR to obtain a partial cDNA clone (squid fiber clone 3, SF3), which we extended in both the 5' and 3' directions to obtain the full-length open-reading frame. The predicted amino-acid sequence of SF3 is similar to sqNDCBE, and a phylogenetic analysis of the membrane domains indicates that SF3 clusters with electroneutral Na(+)-coupled SLC4 transporters. However, when we measure pH(i) and membrane potential--or use two-electrode voltage clamping to measure currents--on Xenopus oocytes expressing SF3, the oocytes exhibit the characteristics of an electrogenic Na/HCO(3)(-) cotransporter, NBCe. That is, exposure to extracellular CO(2)/HCO(3)(-) not only causes a fall in pH(i), followed by a robust recovery, but also causes a rapid hyperpolarization. The current-voltage relationship is also characteristic of an electrogenic NBC. The pH(i) recovery and current require HCO(3)(-) and Na(+), and are blocked by DIDS. Furthermore, neither K(+) nor Li(+) can fully replace Na(+) in supporting the pH(i) recovery. Extracellular Cl(-) is not necessary for the transporter to operate. Therefore, SF3 is an NBCe, representing the first NBCe characterized from an invertebrate.

P‐ERK Activation by Extracellular or Intracellular Acidosis Slows NHE3‐Mediated Acid Extrusion And Activates Ammoniagenesis in LLC‐PK1‐F+ Cells

We exposed proximal tubule-like F+ cells to extracellular acidosis pHo=6.8 or troglitazone (TRO)-induced intracellular acidosis pHi<6.8(pHo=7.4) for 4min and 3h in order to determine cell signaling and physiological responses (NHE3 activity and NH4+production). Exposure to pHo =6.8 for 4min and 3h reduced the pHi from 7.32 ±0.07 to 6.96±0.04 and 6.75±0.11 associated with a 2.3 and 2.7 fold rise in PERK/T-ERK ratio (both p<0.05). Exposure to TRO (20uM) reduced pHi from 7.32±0.07 to 6.60±0.08 and 6.86±0.15 associated with a 3.7 and 2.1 rise in P-ERK at 4min and 3h respectively. NHE3 activity measured after transient 6min exposure to pHo=6.8 was reduced by 32% (p<0.01) while NH4+ increased by 1.65 fold (p<0.05) at 3h; TRO reduced NHE3 activity by 82% and NH4+ 2.4 fold (both p<0.01). TRO-induced P-ERK activation and fall in pHi was prevented by inhibiting EGFR in contrast EGFR inhibition with pHo 6.8 did not decrease P-ERK activation or cellular acidosis. Pre-incubation with MEKK inihibitors PD98059 and UO126 prevented the acidosis-induced increase in P-ERK and ammoniagenesis. Our results are consonant with P-ERK activation associated with extracellular acidosis and intracellular acidosis (K+ deficiency?) playing a major role in regulating acute physiological responses.

PH i Regulation In Central CO 2 ‐Chemosensitive And Non‐Chemosensitive Cells: A Mathematical Model

Numerous studies have suggested that a decrease in intracellular pH (pHi) is the primary stimulus for CO2 sensing in central CO2 chemoreceptors. In CO2 chemosensitive neurons, an increase in CO2 (hypercapnia) leads to a maintained reduction in pHi while in non-chemosensitive neurons pHi recovery is observed. We developed a mathematical model to evaluate the roles of intrinsic buffering capacity, Na+/H+ exchange (NHE), and HCO3−/Cl− exchange (AE) on pHi regulation in response to simulated hypercapnic acidosis. The cell model incorporates conservation of mass and electroneutrality constraints, kinetic models of the Na+/K+-ATPase, AE, NHE3 isoform (believed to play a primary role in CO2 sensing), and passive permeation pathways for ions, nonelectrolytes, and H2O. H+ buffering (both inside and out) is handled by multiple buffer species all subject to the isohydric principle. In response to simulated hypercapnia, the model predicts pHi responses that are consistent with observations in non-chemosensitive cells, including a pHi recovery mechanism that is dependent on NHE (based on simulation of amiloride effects). The model also demonstrates that inhibition of NHE is sufficient to decrease pHi in the absence of increased CO2 and that the pHi fall during simulated hypercapnia is exacerbated. Additional H+ extrusion pathways need to be explored to identify other mechanisms involved in pHi regulation in CO2 chemosensitive neurons. Supported by DK66124

Thrombin stimulation of Cl −/HCO 3− exchange in human platelets

The presence of one acidifying Cl −/HCO 3 − exchange mechanism in human platelets has not been previously reported. This paper demonstrates that this mechanism does function and that it increases its activity after stimulation with thrombin. On resuspension of BCECF-loaded platelets in a chloride-free medium (gluconate replaced) that contains bicarbonate, cytosolic pH (pHi) increased and stabilized after 10 min at an alkaline value. After addition of 50 mM NaCl, pHi fell rapidly reaching steady state in the succeeding 5 min. The stilbene derivative 4-acetamido-4′-isothiocyanato stilbene-2,2′ disulfonic acid (SITS) inhibited both, the alkalization in chloride-poor solution and the recovery from the alkaline load after chloride enrichment. The decline in pHi was observed whether chloride was delivered to the solution in the form of LiCl or NaCl, or when the later was applied after blockage of the Na +/H + exchanger. The recovery in chloride-containing solution was in contrast to the effect of a similar change in osmolarity by addition of 50 mM sodium gluconate that did not produced a significant variation of pHi. Posterior addition of NaCl after 5 min in high gluconate reproduced the pHi fall of the control experiment. Alkali loads produced by 25 mM trimethylamine hydrochloride (TMA) were also counteracted by HCO 3 −-equivalent efflux via Cl −/HCO 3 − exchange. One of the major observations of the present study is that HCO 3 − equivalent efflux was twice as high when the platelets were previously stimulated with 0.1 IU of thrombin, but thrombin did not produce significant changes of the pHi recovery rate in a bicarbonate-free solution. The increase of the decline in pHi elicited by preexposure to thrombin was still observed in the presence of an inhibitor of the Na +/H + exchange or in sodium-free solutions. It is concluded that a Na-independent Cl −/HCO 3 − exchange mechanism mediates the recovery of pHi from alkalosis in platelets and that thrombin activates this exchanger by a direct regulatory pathway.

Early acute necrosis and delayed apoptosis induced by methyl mercury in murine peritoneal neutrophils.

There is growing evidence that heavy metals in general, and mercurial compounds in particular, are immunotoxic. The purpose of this study was to explore the mechanism of MeHg in inducing cell death of mouse peritoneal neutrophils. In this paper we demonstrate that MeHg induces apoptosis and necrosis depending on MeHg concentration. In vitro exposure of mouse peritoneal neutrophils to MeHg resulted in a time- and concentration-dependent cell death. MeHg (15 microM) induced neutrophil necrosis in 13 min. The type of cell death was attributed to necrosis based on cells permeable to the fluorescent dye, propidium iodide and DNA appeared as a smear. With fura-2 microfluorimetric technique, we found that the entry of external Ca2+ into the cytosol played a crucial role in inducing cell necrosis by 15 microM MeHg. However, at lower concentrations, MeHg (10 microM)-induced apoptosis is confirmed by the observation of morphological features characterised by apoptotic bodies and fragmented DNA ladder. MeHg (10 microM) caused an immediate fall in pHi as revealed by the pH-sensitive fluorescent probe 2'7'-bis (carboxyethyl)-5(6)-carboxyfluorescein. We have found that MeHg induced cellular acidification prior to DNA fragmentation so as the other two apoptosis-inducing agents (ZnCl(2) and EGTA). Furthermore, acid-activated endonuclease was increased by MeHg in neutrophils, which we considered to play a possible role in chromatin digestion leading to apoptosis. Taken together, these findings indicate that MeHg induces necrosis at higher concentrations by a rapid increase of [Ca2+]i and apoptosis at lower concentrations by acid activation of endonuclease.