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

Abstract

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.

Keywords: HEPES; acetazolamide; electrophysiology; ion-sensitive microelectrodes; mathematical modeling.

Fig. 1.

Representative experiments showing effects of carbonic anhydrase IV (CA IV) on intracellular pH (pH_i) and surface pH (pH_S) changes evoked by application and removal of CO₂/HCO₃⁻. A: oocytes expressing human CA IV. (dpHi/dt)max, maximal rate of pH_i change; BECF, bulk extracellular fluid. B: oocytes injected only with H₂O. Both in “CA IV” or “H₂O” 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 (see Table 1 in the first paper in this series; ref. 18) and then back again. In this example, the extracellular 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 electrode was withdrawn to the BECF for calibration. At other times the pH_S electrode was dimpling ∼40 μm into the oocyte surface. The left and right blue, dashed vertical lines 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 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. 2.

Summary of effects of CA IV on pH_i and pH_S. This figure summarizes results from a larger number of experiments, such as those in Fig. 1, on oocytes expressing CA IV or injected only with H₂O. 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. This summary includes all oocytes expressing CA IV (and exposed to 1.5% CO₂ in 5 mM HEPES) in the present study, as well as their day-matched H₂O-injected controls. 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. Values are means ± SE, with nos. of oocytes in parentheses. We performed a one-way ANOVA in B and C (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 IV” oocytes was 7.38 ± 0.024 (n = 26) and for “H₂O” oocytes was 7.28 ± 0.025 (n = 19; one-way ANOVA, P = 0.001).

Fig. 3.

Representative experiments showing effects of acetazolamide (ACZ) on pH_i and pH_S. A: oocyte expressing human CA IV. B: H₂O-injected oocyte. The pH_i and pH_S records were obtained from the same oocytes. The extracellular solutions were ND96 at the beginning of the experiment and, during the indicated time, 1.5% CO₂/10 mM HCO₃⁻/pH 7.50 (see Table 1 in the first paper in this series; ref. 18). Both solutions were supplemented, as indicated, with 600 μM ACZ. Control experiments (not shown) on “CA IV” oocytes showed that a second pulse of CO₂/HCO₃⁻, in the absence of ACZ, produced pH_i and pH_S transients that were indistinguishable from the first pulse (n = 4). In both A and B, 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 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 changes in steady-state pH_i. The upward and downward vertical arrows near the pH_S records represent maximal CO₂-induced changes in pH_S (positive or negative direction).

Fig. 4.

Summary of the effects of extracellular ACZ on pH_i and pH_S. This figure summarizes data relating to CO₂ addition and removal from a larger number of experiments, such as those in Fig. 3, on oocytes expressing CA IV or injected with H₂O. After the first assay, during which we switched the extracellular solution from ND96 to 1.5% CO₂/10 mM HCO₃⁻ and then back again, in the absence of ACZ (−ACZ), we exposed the same oocyte to 600 μM ACZ and then repeated the assay (+ACZ). A: maximal rates of pH_i change (negative or positive 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 or negative 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 = 0.0005 in A, P = 0.06 in B, and P < 10⁻⁴ in C), followed by a Student-Newman-Keuls (SNK) analysis (P shown for individual comparisons). The mean initial pH_i for “CA IV” oocytes was 7.37 ± 0.02 (n = 8) and for “H₂O” oocytes was 7.25 ± 0.025 (n = 6; one-way ANOVA, P = 0.046).

Fig. 5.

Representative experiment showing effects of extracellular ACZ on pH_i and pH_S in an oocyte injected with CA II. The pH_i and pH_S records were obtained from the same oocyte. The extracellular solution was ND96 at the beginning of the experiment and, during the indicated time, 5% CO₂/33 mM HCO₃⁻/pH 7.50 (see Table 1 in the first paper in this series; ref. 18). Both solutions were supplemented, as indicated, with 600 μM ACZ. 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 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 changes in steady-state pH_i. The upward and downward vertical arrows near the pH_S records represent maximal CO₂-induced changes in pH_S (positive or negative direction).

Fig. 6.

Summary of the effects of extracellular ACZ in “CA II” oocytes. This figure summarizes data relating to CO₂ addition and removal from a larger number of experiments such as those in Fig. 5. After the first assay, during which we switched the extracellular solution from ND96 to 5% CO₂/33 mM HCO₃⁻ and then back again, in the absence of ACZ (−ACZ), we exposed the same oocyte to 600 μM ACZ and then repeated the assay (+ACZ). A: maximal rates of pH_i change (negative or positive 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 or negative 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 = 0.0008 in A and P = 0.004 in C), followed by a Student-Newman-Keuls (SNK) analysis (P shown for individual comparisons). We performed a paired t-test in B. The average initial pH_i for “CA II” oocytes in the CO₂/HCO₃⁻-free ND96 solution was 7.27 ± 0.02 (n = 6), which is not significantly different from the value reported for “CA II” oocytes in the first paper (18) in this series (7.21 ± 0.05, n = 16; P = 0.52, unpaired t-test).

Fig. 7.

Summary of the effects of injected ACZ in “CA IV” oocytes. This figure summarizes data from experiments (not shown) in which 1) we monitored pH_i and pH_S while exposing oocytes to a solution containing 5% CO₂/33 mM HCO₃⁻ (Pre-ACZ injection) and then withdrew the CO₂/HCO₃⁻; 2) we removed the oocytes from the chamber and injected them with ACZ, and, after ∼3 h, 3) we repeated the electrophysiological assay (Post-ACZ injection). A: maximal rates of pH_i change (negative direction) produced by the extracellular addition of CO₂/HCO₃⁻. 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 addition of CO₂/HCO₃⁻. 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 = 0.004 in B, and P < 10⁻⁴ in C), followed by a Student-Newman-Keuls (SNK) analysis (P shown for individual comparisons). The average initial pH_i in the CO₂/HCO₃⁻-free ND96 solution was 7.39 ± 0.01 (n = 7) for “CA IV” oocytes, 7.21 ± 0.01 (n = 4) for “H₂O” oocytes, and 7.24 ± 0.03 (n = 5) for “CA II” oocytes.

Fig. 8.

Summary of time constants (τ) for pH_i and pH_S changes for a larger number of experiments, such as those in Fig. 1, on oocytes expressing CA IV or injected with H₂O. Values are means ± SE, with nos. of oocytes in parentheses. We performed a one-way ANOVA (overall P value: P < 10⁻⁴) followed by a Student-Newman-Keuls (SNK) analysis (P shown for individual comparisons).

Fig. 9.

Representative experiments showing the effect of graded increases in extracellular CO₂/HCO₃⁻ levels on pH_i and pH_S. A: oocyte expressing CA IV. B: oocyte injected with H₂O. A and B each represent an experiment on a single oocyte. 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 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 electrode was moved 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 expressing CA IV or injected with H₂O. 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 (overall P values: P < 10⁻⁴ in A, in B, and in C) followed by a Student-Newman-Keuls (SNK) analysis (P shown for individual comparisons). For all experiments, the solution flowed at 3 ml/min and the sampling rate was 1 per 500 ms.

Fig. 11.

Summary of time constants (τ) for pH_i and pH_S changes for a larger number of experiments, such as those in Fig. 10, on oocytes expressing CA IV or injected with H₂O. Values are means ± SE, with nos. of oocytes in parentheses. We performed a one-way ANOVA (overall P < 10⁻⁴) followed by a Student-Newman-Keuls (SNK) analysis (P shown for individual comparisons).

Fig. 12.

Representative experiments showing the effect of 1.5% CO₂/10 mM HCO₃⁻ with graded increases in HEPES levels on pH_i and pH_S. A: oocyte expressing CA IV. B: oocyte injected with H₂O. A and B each represent an experiment on a single oocyte. 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) CO₂/HCO₃⁻ + 1 mM HEPES, or 2) CO₂/HCO₃⁻ + 5 mM HEPES, or 3) CO₂/HCO₃⁻ + 25 mM HEPES (see Table 1 in the first paper in this series; ref. 18). 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 electrode was moved 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 the CO₂-induced change in steady-state pH_i. The upward vertical arrows represent the maximal excursion of pH_S (positive direction).

Fig. 13.

Summary of the effect of graded increases in extracellular HEPES levels on pH_i and pH_S. This figure summarizes results from a larger number of experiments, such as those in Fig. 12, A and B. A and E: maximal rates of pH_i change (negative direction) produced by the introduction of extracellular CO₂/HCO₃⁻. B and F: changes in steady-state pH_i induced by the addition of CO₂/HCO₃⁻. C and G: maximal changes in pH_S (positive direction) caused by the addition of CO₂/HCO₃⁻. D and H: time constants for pH_S decay (τ_pHS) caused by the addition of CO₂/HCO₃⁻. Values are means ± SE, with nos. of oocytes in parentheses. We performed a one-way ANOVA (overall P values: P < 10⁻⁴ for A with E, B with F, and C with G, and P = 0.0009 for D with H) followed by a Student-Newman-Keuls (SNK) analysis (P shown for individual comparisons). For all experiments, the solution flowed at 3 ml/min and the sampling rate was 1 per 500 ms.

Fig. 14.

Representative experiments showing the effect of 1.5% CO₂/10 mM HCO₃⁻ with graded increases in HEPES levels (+ extracellular bovine CA II = 0.1 mg/ml) on pH_i and pH_S. A: oocyte expressing CA IV. B: oocyte injected with H₂O. A and B each represent an experiment on a single oocyte. The pH_i trace is represented by the red lower record, and pH_S, by the green upper record. At the indicated time, we switched the extracellular (ex) solution from ND96 to 1) CO₂/HCO₃⁻ + 1 mM HEPES, or 2) CO₂/HCO₃⁻ + 5 mM HEPES, or 3) CO₂/HCO₃⁻ + 25 mM HEPES (see Table 1 in the first paper in this series; ref. 18). 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 electrode was moved 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 the CO₂-induced change in steady-state pH_i. The upward vertical arrows represent the maximal excursion of pH_S (positive direction).

Fig. 15.

Summary of the effect of graded increases in extracellular HEPES levels on pH_i and pH_S (+ extracellular bovine CA II = 0.1 mg/ml). This figure summarizes results from a larger number of experiments, such as those in Fig. 14, A and B. A and E: maximal rates of pH_i change (negative direction) produced by the introduction of extracellular CO₂/HCO₃⁻. B and F: changes in steady-state pH_i induced by the addition of CO₂/HCO₃⁻. C and G: maximal changes in pH_S (positive direction) caused by the addition of CO₂/HCO₃⁻. D and H: time constants for pH_S decay (τ_pHS) caused by the addition of CO₂/HCO₃⁻. Values are means ± SE, with nos. of oocytes in parentheses. We performed a one-way ANOVA (overall P values: P < 10⁻⁴ for A with E, B with F, C with G, and D with H) followed by a Student-Newman-Keuls (SNK) analysis (P shown for individual comparisons). For all experiments, the solution flowed at 3 ml/min and the sampling rate was 1 per 500 ms.

Fig. 16.

Representative experiments showing the effect of 10% CO₂/66 mM HCO₃⁻ with graded increases in HEPES levels on pH_i and pH_S. A: oocyte expressing CA IV. B: oocyte injected with H₂O. A and B each represent an experiment on a single oocyte. The pH_i trace is represented by the red lower record, and pH_S, by the green upper record. At the indicated time, we switched the extracellular solution from ND96 to 1) CO₂/HCO₃⁻ + 1 mM HEPES, or 2) CO₂/HCO₃⁻ + 5 mM HEPES, or 3) CO₂/HCO₃⁻ + 25 mM HEPES (see Table 1 in the first paper in this series; ref. 18). 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 electrode was moved 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 the CO₂-induced change in steady-state pH_i. The upward vertical arrows represent the maximal excursion of pH_S (positive direction).

Fig. 17.

Summary of the effect of graded increases in extracellular HEPES levels on pH_i and pH_S. This figure summarizes results from a larger number of experiments, such as those in Fig. 16, A and B. A and E: maximal rates of pH_i change (negative direction) produced the introduction of extracellular CO₂/HCO₃⁻. B and F: changes in steady-state pH_i induced by the addition of CO₂/HCO₃⁻. C and G: maximal changes in pH_S (positive direction) caused by the addition of CO₂/HCO₃⁻. D and H: time constants for pH_S decay (τ_pHS) caused by the addition of CO₂/HCO₃⁻. Values are means ± SE, with nos. of oocytes in parentheses. We performed a one-way ANOVA (overall P values: P < 10⁻⁴ for A with E, B with F, C with G, and D with H) followed by a Student-Newman-Keuls (SNK) analysis (P shown for individual comparisons). For all experiments, the solution flowed at 3 ml/min and the sampling rate was 1 per 500 ms.

Fig. 18.

Studies with dual pH_S electrodes. A and B: representative experiments on an oocyte expressing CA IV (A) and an oocyte injected with H₂O (B). The inset is a schematic top view of the oocyte (darker animal pole facing upward), showing the arrangement of microelectrodes; the red arrows show the direction of convective flow. The tip of electrode no. 1 is ∼5° downstream from the meridian perpendicular to the direction of bulk flow, and at the equator (border between the upward facing animal pole and downward facing vegetal pole of the oocyte); its pH_S record is green. The tip of electrode no. 2 is at the back of the oocyte; its pH_S record is magenta. In these experiments, the solution flowed at 3 ml/min and the sampling rate was 1 per 500 ms. C–E: summary of experiments with dual pH_S electrodes. These panels summarize results from a larger number of experiments, such as those in A and B. C shows time to peak for pH_S produced by the introduction of extracellular CO₂/HCO₃⁻. D shows maximal changes in pH_S (positive direction) caused by the addition of CO₂/HCO₃⁻. E shows time constants for pH_S decay (τ_pHS) caused by the addition of CO₂/HCO₃⁻. Values are means ± SE, with nos. of oocytes in parentheses. We performed a one-way ANOVA (overall P values: P = 0.00013 in C, P = 10⁻⁴ in D, and P < 10⁻⁴ in E) followed by a Student-Newman-Keuls (SNK) analysis (P shown for individual comparisons). For all experiments, the solution flowed at 3 ml/min and the sampling rate was 1 per 500 ms.

Fig. 19.

Predictions of the mathematical model for concentration-time profiles of CO₂ for a “CA IV” oocyte (red curves) and a “Control” or “H₂O” oocyte (brown curves). A: [CO₂] near the intracellular surface of the plasma membrane (IM). B: [CO₂] near 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. Figure 20 illustrates the dimensions of the IM and EM regions. In modeling the “CA IV” oocyte, we implemented a large increase in CA activity on the extracellular surface of the oocyte, and a small increase in the cytosol. Details of the mathematical model are presented in the third paper in this series (21).

Fig. 20.

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 IV” oocyte. B: “Control” or “H₂O” oocyte. B is identical to Fig. 12B in the first paper in this series (18). 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 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 (21).

Fig. 21.

Comparison of physiological τ data with t₆₃ data predicted by the mathematical model. The physiological data are taken from Fig. 8; note that here we report the means ± standard deviation (and not SE, as in Fig. 8). The simulated “Control” data are identical to those in Fig. 13 in the first paper in this series (18). 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 IV and control conditions. The inconsistency probably arises, in part, because of the multiexponential decay of the simulated pH_S 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 (21).

Fig. 22.

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 IV” oocyte. B: “Control” or “H₂O” oocyte. B is identical to Fig. 14B in the first paper in this series (18). 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 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 the series (21).

Fig. 23.

Comparison of physiological data with data predicted by the mathematical model during addition of 1.5% CO₂/10 mM HCO₃⁻ with graded increases in HEPES levels. A and D: maximal rates of pH_i change (negative direction) produced by the introduction of extracellular CO₂/HCO₃⁻. B and E: maximal changes in pH_S (positive direction) caused by the addition of CO₂/HCO₃⁻. C and F: time constants for pH_S decay in physiological experiments (τ_pHS) and time to 63% of completion (i.e., t₆₃) for pH_S decay predicted by mathematical model during the addition of CO₂/HCO₃⁻. The physiological data are taken from Fig. 13; note that here we report the means ± standard deviation (and not SE, as in Fig. 13). Details of the mathematical model are presented in the third paper in this series (21).

Fig. 24.

Comparison of physiological data with data predicted by the mathematical model during addition of 10% CO₂/66 mM HCO₃⁻ with graded increases in HEPES levels. A and D: maximal rates of pH_i change (negative direction) produced by the introduction of extracellular CO₂/HCO₃⁻. B and E: maximal changes in pH_S (positive direction) caused by the addition of CO₂/HCO₃⁻. C and F: time constants for pH_S decay (τ_pHS) in physiological experiments and time to 63% of completion (i.e., t₆₃) for pH_S decay predicted by mathematical model during the addition of CO₂/HCO₃⁻. The physiological data are taken from Fig. 17; note that here we report the means ± standard deviation (and not SE, as in Fig. 17). Details of the mathematical model are presented in the third paper in this series (21).