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

Abstract

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.

Keywords: electrode depth; electrophysiology; ethoxzolamide; ion-sensitive microelectrodes; mathematical modeling.

Fig. 1.

Model of an oocyte exposed to CO₂/HCO₃⁻. The main part of the figure illustrates the diffusion and reaction events as CO₂ enters an oocyte. The black arrows with sharp heads indicate solute diffusion in the extracellular unconvected fluid (EUF) and intracellular fluid. The black arrows with dull heads indicate reactions. The left inset is a schematic top view of the oocyte in the chamber, as seen through the microscope. The orange arrows indicate the direction of convective flow of the bulk extracellular fluid (BECF). The right inset is a schematic view of the oocyte along the axis of convective flow, looking downstream. The darker half of the oocyte, oriented upward, is the animal pole. CA II, carbonic anhydrase II; pH_i, intracellular pH; pH_S, surface pH.

Fig. 2.

Representative experiments showing effects of CA II on pH_i and pH_S changes evoked by application and removal of CO₂/HCO₃⁻. A: oocytes injected with recombinant human CA II dissolved in Tris buffer. B: oocytes injected only with Tris buffer. Both in “CA II” or “Tris” oocytes, the pH_i trace is represented by the red lower record, and pH_S, by the green upper record. At the indicated times, we switched the extracellular solution from ND96 to 1.5% CO₂/10 mM HCO₃⁻/pH 7.50 (Table 1) and then back again. In this example, the extracellular solution flowed at 2 ml/min and the sampling rate was 1 per 1,000 ms. The vertical gray bands represent periods during which the pH_S electrode was withdrawn to the BECF for calibration. At other times the pH_S electrode was dimpling ∼40 μm into the oocyte surface. The gray, dashed vertical lines represent the times that the computer switched the valves to initiate a change of solutions. The left and right blue, dashed vertical lines (see results) represent the initiation of the pH_S and pH_i transients, respectively. The dashed black lines through the initial portions of the pH_i records for CO₂ application and removal represent best linear fits for maximal rates of pH_i change (negative or positive direction). The downward vertical arrows near the pH_i records represent the CO₂-induced changes in steady-state pH_i. The upward and downward arrows near the pH_S records represent maximal changes in pH_S (positive or negative direction).

Fig. 3.

Summary of effects of CA II on pH_i and pH_S. This figure summarizes results from a larger number of experiments, such as those in Fig. 2, on oocytes injected with CA II in Tris buffer, or only Tris buffer. In each case, we switched the extracellular solution from ND96 to 1.5% CO₂/10 mM HCO₃⁻ (gray bars) and vice versa (white bars). A: maximal rates of pH_i change (negative or positive direction) produced by the extracellular solution switch. B: changes in steady-state pH_i, induced by the addition of CO₂/HCO₃⁻. C: maximal changes in pH_S (positive or negative direction) caused by the extracellular solution switch. This summary includes all oocytes injected with CA II (and exposed to 1.5% CO₂ in 5 mM HEPES) in the present study, as well as their day-matched Tris-injected controls. Values are means ± SE, with nos. of oocytes in parentheses. We performed a one-way ANOVA (overall P values: P < 10⁻⁴ in A, and P < 10⁻⁴ in C), followed by a Student-Newman-Keuls (SNK) analysis (P shown for individual comparisons). We performed a paired t-test in B. The mean initial pH_i for “CA II” oocytes in the CO₂/HCO₃⁻-free ND96 solution was 7.21 ± 0.05 (n = 16). This mean is not significantly different from that for “Tris” oocytes, 7.22 ± 0.03 (n = 16; P = 0.93).

Fig. 4.

Representative experiments showing effects of ethoxzolamide (EZA) EZA on pH_i and pH_S. A: oocyte injected with recombinant human CA II dissolved in Tris buffer, assayed before treatment with EZA (Pre-EZA). B: “CA II” oocyte, assayed after a 3-h treatment with EZA (Post-EZA). C: oocyte injected only with Tris buffer, assayed before treatment with EZA. D: “Tris” oocyte, assayed after a 3-h treatment with EZA. For both CA II and Tris experiments, the Pre-EZA and Post-EZA pH_i and pH_S records were obtained from the same oocytes. Between the two CO₂/HCO₃⁻ exposures, the oocytes were exposed for 3 h to the ND96 solution (Table 1) supplemented with 400 μM EZA, followed by 3 washes in ND96, and incubation in ND96 (30 min to 3 h). We noticed no effect of the variable duration of the post-EZA incubation. The extracellular solutions were ND96 and, during the indicated time, 1.5% CO₂/10 mM HCO₃⁻/pH 7.50. In both experiments, the solution flowed at 2 ml/min and the sampling rate was 1 per 1,000 ms. The vertical gray bands represent periods during which the pH_S electrode was withdrawn to the BECF for calibration. The dashed black lines through the initial portions of the pH_i records for CO₂ application and removal represent best linear fits for maximal rates of pH_i change (negative or positive direction). The downward vertical arrows represent CO₂-induced change in steady-state pH_i. The upward and downward vertical arrows near the pH_S records represent the maximal CO₂-induced changes in pH_S (positive or negative direction).

Fig. 5.

Summary of the effects of EZA on pH_i and pH_S. This figure summarizes data relating to CO₂ addition from a larger number of experiments, such as those in Fig. 4, on oocytes injected with CA II in Tris buffer, or only Tris buffer. After an assay during which we switched the extracellular solution from ND96 to 1.5% CO₂/10 mM HCO₃⁻, in the absence of EZA (Pre-EZA), we exposed the same oocyte to 400 μM EZA for 3 h, and then repeated the assay (Post-EZA). A: maximal rates of pH_i change (negative direction) produced by the extracellular solution switch. B: changes in steady-state pH_i produced by the switch to CO₂/HCO₃⁻. C: maximal changes in pH_S (positive direction) produced by the extracellular solution switch. Values are means ± SE, with nos. of oocytes in parentheses. We performed a one-way ANOVA (overall P values: P < 10⁻⁴ in A, P = 0.38 in B, and P = 0.0012 in C), followed by a Student-Newman- Keuls (SNK) analysis (P shown for individual comparisons). The mean initial pH_i for “CA II” oocytes in the CO₂/HCO₃⁻-free ND96 solution was 7.13 ± 0.11 (n = 6), which is not significantly different from the mean value for “Tris” oocytes, 7.05 ± 0.07 (n = 5; P = 0.93). For both “CA II” and “Tris” oocytes, EZA had no effect on the initial pH_i.

Fig. 6.

Summary of time constants (τ) for pH_i and pH_S changes for a larger number of experiments, such as those in Fig. 2, on oocytes injected with CA II in Tris buffer, or only Tris. Values are means ± SE, with nos. of oocytes in parentheses. We performed a one-way ANOVA followed by a Student-Newman-Keuls (SNK) analysis (P shown for individual comparisons).

Fig. 7.

Representative experiments with dual pH_i-electrode impalements of Xenopus oocytes. A: oocyte injected with H₂O on day 0. B: oocyte injected with recombinant human CA II dissolved in Tris buffer on day 3. The inset is a schematic top view of the oocyte, showing the arrangement of microelectrodes; the orange arrows show the direction of convective flow. C: oocyte injected only with Tris buffer on day 3. The extracellular solutions were ND96 (Table 1) and, during the indicated time, 1.5% CO₂/10 mM HCO₃⁻/pH 7.50. For all three oocytes, the solution flowed at 3 ml/min and the sampling rate was 1 per 500 ms. The pH_i record from electrode no. 1 (superficial) is in red, and the pH_i record from electrode no. 2 (deep) is in purple. The gray, dashed vertical lines represent the times that the computer switched the valves to initiate a change of solutions. The left and right blue, dashed vertical lines represent the initiation of the transients for pH_i electrodes no. 1 and no. 2, respectively. The dashed black lines through the initial portions of the pH_i records for CO₂ application represent best linear fits for maximal rates of pH_i change (negative direction). The downward vertical arrows represent the CO₂-induced change in steady-state pH_i.

Fig. 8.

Summary of dual pH_i-electrode impalements. This figure summarizes results from a larger number of experiments, such as those in Fig. 7. We injected all oocytes with H₂O on day 0. Those for which this was the only injection are labeled “H₂O.” We impaled these oocytes superficially (S) with two pH_i microelectrodes, electrode no. 1 positioned by a manual manipulator (the one that held pH_i electrodes in other protocols), and electrode no. 2 positioned by a motorized manipulator (the one that held pH_S electrodes in other protocols). For other oocytes, we followed the injection of water on day 0 with the injection on day 3 with either “CA II” in Tris buffer, or only “Tris.” We impaled these oocytes superficially with pH_i microelectrode no. 1, and deep (D) with electrode no. 2. A: time lags between initiation of the transients for pH_i electrodes no. 1 and no. 2. The hatched bars represent the oocyte-by-oocyte difference between electrodes no. 2 and no. 1. An ANOVA for the six differences in time lags between electrode no. 2 and electrode no. 1 (six hatched bars) had an overall P value of 0.0003. The two “H₂O” values were significantly different from the others. B: maximal rates of pH_i change (negative or positive direction) produced by the extracellular solution switch. C: time constants (τ) for pH_i changes. D: changes in steady-state pH_i induced by the addition of CO₂/HCO₃⁻. Values are means ± SE, with nos. of oocytes in parentheses. We performed a one-way ANOVA, followed by a Student-Newman-Keuls (SNK) analysis (P shown for individual comparisons). The initial pH_i values for “H₂O”, “CA II”, and “Tris” oocytes in the ND96 (i.e., CO₂/HCO₃⁻-free) solution were not significantly different from one another, both for electrode no. 1 and electrode no. 2 (overall ANOVA, P = 0.81).

Fig. 9.

Representative experiments showing the effect of graded increases in extracellular CO₂/HCO₃⁻ levels on pH_i and pH_S. A: oocyte injected with recombinant human CA II in Tris buffer. B: oocyte injected only with Tris buffer. A and B each represent an experiment on single oocytes. The pH_i trace is represented by the red lower record, and pH_S, by the green upper record. At the indicated times, we switched the extracellular solution (Table 1) from 1) ND96 to 1.5% CO₂/10 mM HCO₃⁻ (left), or 2) ND96 to 5% CO₂/33 mM HCO₃⁻ (center), or 3) ND96 to 10% CO₂/66 mM HCO₃⁻ (right). After the first two CO₂/HCO₃⁻ exposures, we restored the ND96 solution (not shown). For both oocytes, the solution flowed at 3 ml/min and the sampling rate was 1 per 500 ms. The vertical gray bands represent periods during which the pH_S electrodes were withdrawn to the BECF for calibration. The dashed black lines through the initial portions of the pH_i records for CO₂ application represent best linear fits for maximal rates of pH_i change (negative direction). The downward vertical arrows represent CO₂-induced changes in steady-state pH_i. The upward vertical arrows represent the maximal excursion of pH_S (positive direction).

Fig. 10.

Summary of effects of graded increases of extracellular CO₂/HCO₃⁻ levels on pH_i and pH_S. This figure summarizes results from a larger number of experiments, such as those in Fig. 9, on oocytes injected with CA II in Tris buffer, or only Tris buffer. A: maximal rates of pH_i change (negative direction) produced by the introduction of extracellular CO₂/HCO₃⁻. B: changes in steady-state pH_i induced by the addition of CO₂/HCO₃⁻. C: maximal changes in pH_S (positive direction) caused by the addition of CO₂/HCO₃⁻. Values are means ± SE, with nos. of oocytes in parentheses. The white numerals are the ratios of mean values, relative to the value at 1.5% CO₂. We performed a one-way ANOVA followed by a Student-Newman-Keuls (SNK) analysis (P shown for individual comparisons). The initial pH_i values for “CA II” or “Tris” oocytes in the ND96 (i.e., CO₂/HCO₃⁻-free) solution were not significantly different (overall ANOVA, P = 0.84).

Fig. 11.

Predictions of the mathematical model for concentration-time profiles of CO₂ for a “CA II” oocyte (red curves) and a “Control” or “Tris” oocyte (brown curves). A: [CO₂] at the intracellular surface of the plasma membrane (IM). B: [CO₂] at the extracellular surface of the plasma membrane (EM). C: gradient of CO₂ across the plasma membrane (PM), the result of subtracting values in B from corresponding values in A. The hatched areas in A and B identify the initial phase of CO₂ influx, during which the model predicts that cytosolic CA decreases [CO₂] at the inner and outer surfaces of the plasma membrane. However, the dominant effect is at the inner membrane, so that CA provides a larger gradient for CO₂ influx, as indicated by the hatched area in C. Details of the mathematical model are presented in the third paper in this series (49).

Fig. 12.

Predictions of the mathematical model for maximal CO₂ diffusion and reaction fluxes near the plasma membrane during addition of 1.5% CO₂/10 mM HCO₃⁻. A: “CA II” oocyte. B: “Control” or “Tris” oocyte. The parallel dark brown curves identify the plasma membrane (PM). The green curves to the left and right of the PM represent the spatial discretizations for the numerical solutions. The numbers near the arrows 1 through 5 indicate the maximal diffusive fluxes (sharp arrowheads) and reaction fluxes (dull arrowheads) in units of μmol/s. These maximal fluxes occur within ∼1 s of each other. The diffusion/reaction ratio (DRR) is the quotient of flux no. 1/flux no. 4. Details of the mathematical model are presented in the third paper in this series (49).

Fig. 13.

Comparison of physiological τ data with t₆₃ data predicted by the mathematical model. The physiological data are taken from Fig. 6; note that here we report the means ± standard deviation (and not SE, as in Fig. 6). An apparent inconsistency arises when we compare pH_S data from physiological experiments (time constant, τ) and simulations (time to 63% of completion, t₆₃) for CO₂ addition, under both CA II and control conditions. The inconsistency probably arises, in part, because of the multiexponential decay of the simulated pH trajectory, the special nature of the pH_S trajectory beneath the pH_S electrode, and the asymmetry of pH_S over the oocyte surface. Details of the mathematical model are presented in the third paper in this series (49).

Fig. 14.

Predictions of the mathematical model for maximal CO₂ diffusion and reaction fluxes near the plasma membrane during removal of 1.5% CO₂/10 mM HCO₃⁻. A: “CA II” oocyte. B: “Control” or “Tris” oocyte. The parallel dark brown curves identify the plasma membrane (PM). The green curves to the left and right of the PM represent the spatial discretizations for the numerical solutions. The numbers near the arrows 1 through 5 indicate the maximal diffusive fluxes (sharp arrowheads) and reaction fluxes (dull arrowheads) in units of μmol/s. These maximal fluxes occur within ∼1 s of each other. The diffusion/reaction ratio (DRR) is the quotient of flux no. 1/flux no. 4. Details of the mathematical model are presented in the third paper in this series (49).

Fig. 15.

Predictions of the mathematical model for different depths of impalement, during the application of 1.5% CO₂/10 mM HCO₃⁻ to a “Control” or “Tris” oocyte. We report pH_i trajectories for the first 60 s for six depths (∼50, 100, 150, 200, 250, and 300 μm) for a hypothetical oocyte having a radius of 650 μm. At progressively greater depths, pH_i remains relatively stable for progressively longer time periods before beginning the transition to a near-linear phase of decline. Details of the mathematical model are presented in the third paper in this series (49).

Fig. 16.

Predictions of the mathematical model for pH_i trajectories at depths of ∼50 μm and ∼300 μm. A: simulations for a “CA II” oocyte. B: simulations for a “Control” or “Tris” oocyte. In these simulations, which are comparable to the physiological experiments in Fig. 7, the tortuosity factor (λ) used to simulate a layer of intracellular vesicles is ∼3.16. Details of the mathematical model are presented in the third paper in this series (49).

Fig. 17.

Comparison of predictions of the model, at depths of ∼50 μm (S, Superficial) and ∼300 μm (D, Deep), and the physiological data. A: difference (Deep-Superficial) between pH_i time lags. B: (dpH_i/dt)_max values. C: time constants from physiological experiments or time to 63% of completion (i.e., t₆₃) for simulations. The gray background identifies data for “CA II” oocytes; the absence of background color identifies “Control” or “Tris” oocytes. The physiological data are taken from Fig. 8; note that here we report means ± standard deviation (and not SE, as in Fig. 8).