Transport of H2S and HS(-) across the human red blood cell membrane: rapid H2S diffusion and AE1-mediated Cl(-)/HS(-) exchange

Abstract

The rates of H2S and HS(-) transport across the human erythrocyte membrane were estimated by measuring rates of dissipation of pH gradients in media containing 250 μM H2S/HS(-). Net acid efflux is caused by H2S/HS(-) acting analogously to CO2/HCO3(-) in the Jacobs-Stewart cycle. The steps are as follows: 1) H2S efflux through the lipid bilayer and/or a gas channel, 2) extracellular H2S deprotonation, 3) HS(-) influx in exchange for Cl(-), catalyzed by the anion exchange protein AE1, and 4) intracellular HS(-) protonation. Net acid transport by the Cl(-)/HS(-)/H2S cycle is more efficient than by the Cl(-)/HCO3(-)/CO2 cycle because of the rapid H2S-HS(-) interconversion in cells and medium. The rates of acid transport were analyzed by solving the mass flow equations for the cycle to produce estimates of the HS(-) and H2S transport rates. The data indicate that HS(-) is a very good substrate for AE1; the Cl(-)/HS(-) exchange rate is about one-third as rapid as Cl(-)/HCO3(-) exchange. The H2S permeability coefficient must also be high (>10(-2) cm/s, half time <0.003 s) to account for the pH equilibration data. The results imply that H2S and HS(-) enter erythrocytes very rapidly in the microcirculation of H2S-producing tissues, thereby acting as a sink for H2S and lowering the local extracellular concentration, and the fact that HS(-) is a substrate for a Cl(-)/HCO3(-) exchanger indicates that some effects of exogenous H2S/HS(-) may not result from a regulatory role of H2S but, rather, from net acid flux by H2S and HS(-) transport in a Jacobs-Stewart cycle.

Keywords: AE1; erythrocyte; hydrogen sulfide; sulfide anion; transport.

Fig. 1.

A: Jacobs-Stewart cycle for net efflux of acid. Starting at the top and going clockwise, the steps are as follows: 1) Cl⁻/HCO₃⁻ exchange, 2) intracellular HCO₃⁻ protonation and dehydration, 3) CO₂ efflux, and 4) extracellular CO₂ hydration and deprotonation. B: Jacobs-Stewart cycle for net efflux of acid mediated by Cl⁻/HS⁻ exchange, intracellular HS⁻ protonation, H₂S efflux, and extracellular H₂S deprotonation. C: net result of 1 cycle.

Fig. 2.

Effect of ethoxzolamide on rate of pH equilibration in human erythrocytes at 37°C and atmospheric CO₂. Washed erythrocytes (1.75 ml) were suspended in 160 mM KCl buffered with 1 mM glycylglycine and 0.5 mM HEPES. Extracellular buffer power was 14 μmol/pH, and intracellular buffer power was 115 μmol/pH. A: pH vs. time. Each upstroke of the pH trace represents addition of 5 μmol of NaOH, and each downstroke represents addition of 5 μmol of HCl. Ethoxzolamide (EthZ, 10 μM) was added from a 10 mM stock solution in ethanol at each arrow. Final ethanol concentration was 0.2%. B: rate constants for pH equilibration calculated from pH transients over the range 0.07–0.02 unit from the final value. Point with error bars at right represents mean and range of rates of CO₂-independent pH equilibration at pH 7.3–7.5 previously published (29) corrected to 37°C and the extracellular buffer power in the present experiment. Rate of pH equilibration in the presence of 20 μM ethoxzolamide is approximately equal to the CO₂-independent rate.

Fig. 3.

Stimulation of pH equilibration by addition of Na₂S. Continuation of the experiment in Fig. 2. A: pH vs. time, with the last acid and base pulse in Fig. 2 repeated. Two upstrokes labeled Na₂S represent addition of 5 μmol of Na₂S from a 0.1 M stock solution that had been frozen until use. Other upstrokes and downstrokes represent additions of 5 μmol of NaOH and HCl, respectively. H₂DIDS was added from a 5 mM stock solution to a final concentration of 10 μM. B: rate constants for pH equilibration calculated from the pH transients as described in Fig. 2 legend. In 4 experiments performed at equilibrium pH 7.35–7.45, the mean Na₂S-stimulated rate constant for pH equilibration was 4.5 min⁻¹ (range 3.9–5.5), measured immediately after the Na₂S pulse.

Fig. 4.

Rate constants for pH equilibration following NaOH or HCl pulses at equilibrium pH 8.4–8.45, 37°C, and atmospheric CO₂ and with 160 mM KCl buffered with 1 mM glycylglycine and 0.5 mM HEPES. The first gray bar following the bracket labeled 250 μM H₂S/HS⁻ is the rate constant following a pulse of 5 μmol of Na₂S rather than NaOH.

Fig. 5.

Lack of stimulation of pH equilibration by Na₂SO₃. Cells were suspended in weakly buffered 160 mM KCl as described previously, and 20 μM ethoxzolamide was added to inhibit CO₂-dependent pH equilibration. The number adjacent to each pH transient is the rate constant (min⁻¹) for relaxation of the transient. Each arrow represents addition of 5 μmol of the indicated acid, base, or salt. Na₂SO₃ does not cause as large a pH transient as Na₂S, because SO₃²⁻ is not as strong a base as S²⁻. Rate constant for relaxation of the transient could nonetheless be measured and is not detectably different from the rate constant following NaOH. As described previously, Na₂S stimulates the rate of pH equilibration by ∼5 min⁻¹, with the rate decaying with subsequent acid and base additions.

Fig. 6.

Rate constant (min⁻¹) for sulfide-dependent pH equilibration predicted by the Jacobs-Stewart cycle plotted vs. rate constant k_ex (s⁻¹) for Cl⁻/HS⁻ exchange for various values of the H₂S permeability coefficient P_H2S (cm/s). Predicted value of k_pH was calculated as described in Modeling, with extracellular buffer power one-eighth of intracellular buffer power. Gray zone represents range of values of sulfide-dependent k_pH at equilibrium pH 7.3–7.5 for the same buffer conditions (k_pH = 3.9–5.5 min⁻¹, 4 experiments). If P_H2S is >0.001 cm/s, the observed rate is mainly limited by Cl⁻/HS⁻ exchange and requires a Cl⁻/HS⁻ exchange rate constant ≥2.5 s⁻¹. If P_H2S is <0.0003 cm/s, even a very high Cl⁻/HS⁻ exchange rate could not explain the k_pH at pH 7.4, because the cycle would be limited by H₂S transport.

Fig. 7.

Relative Cl⁻/X⁻ exchange rates for various inorganic and organic anions via mammalian AE1. Present data indicate that the Cl⁻/HS⁻ exchange rate is about one-third that of Cl⁻ and HCO₃⁻ and is comparable to that of Cl⁻/Br⁻ exchange (19, 20, 64) and Cl⁻/formate exchange (23, 30). F⁻, I⁻ (64), oxalate (Ox²⁻), glycolate (Glyc⁻), glyoxalate (Glyox⁻) (30), phosphate, phosphite, hypophosphite (16), acetate (Ac⁻) (18, 20), and sulfate (47) exchange for Cl⁻ much more slowly.