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.

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

“…We selected stage V-VI oocytes and kept them at 18°C in sterile-filtered OR3 medium, each 2 liters of which contained one pack of powdered Leibovitz L-15 media with L-glutamine (GIBCO-BRL), 100 ml of 10,000 U/ml penicillin, 10,000 U/ml streptomycin solution (SigmaAldrich), and 5 mM HEPES titrated to pH 7.50, and osmolality adjusted to 195 mosmol/kgH 2O. One day after their isolation, we injected the oocytes with 50 nl of H2O as a control for the injection of cRNA encoding CA IV in the accompanying paper (46). Three days after this H2O injection, we injected the oocytes with 50 nl of fluid, either Tris buffer (50 mM titrated to pH 7.5 with HCl) or Tris buffer containing 6 ng/nl of recombinant human CA II protein (i.e., 300 ng/oocyte Х 10 pmol, for a final [CA II] i of ϳ30 M, ϳ50% higher than the level in red blood cells).…”

“…control for experiments in the second paper in this series (46), in which, on day 0, we injected cRNA encoding CA IV. 2) After waiting 3 days, we randomly divided the oocytes into two groups (day 3/step b).…”

“…The purpose of the present study, summarized in this paper and its two companions (46,49), is to elucidate the influence of CAs and non-CO 2 /HCO 3 Ϫ buffers on CO 2 fluxes across the plasma membranes of living cells. 1 In 1984, De Hemptinne and Huguenin (31) used a miniaturized electrode, with a glass sensor, to monitor the pH at the extracellular surface (pH S ) of rat soleus muscle as they applied CO 2 /HCO 3 Ϫ .…”

“…54). In the second paper (46), we examine the effect of expressing CA IV (GPI-linked to the extracellular cell surface), with and without CA II in the bulk extracellular fluid (BECF). Early exploratory work by Nakhoul et al (48) on the heterologous expression of CA IV in oocytes motivated portions of this project.…”

How carbonic anhydrases and pH buffers facilitate the movement of carbon dioxide through biological membranes. Focus on “Evidence from simultaneous intracellular- and surface-pH transients that carbonic anhydrase II enhances CO2 fluxes across Xenopus oocyte plasma membranes”; “Evidence from simultaneous intracellular- and surface-pH transients that carbonic anhydrase IV enhances CO2 fluxes across Xenopus oocyte plasma membranes”; and “Evidence from mathematical mo

“…They also demonstrated, as originally shown by Gutknecht and Tosteson (3), that, in the presence of CA, addition of an organic pH buffer, such as HEPES, also accelerates CO 2 Influx. Furthermore, as first shown by Gutknecht and Tosteson, addition of CA II in the bulk extracellular solution also sped up CO 2 influx of both water-and CA IV-injected oocytes (6). This can also be easily explained: addition of a buffer constitutes a novel source of H ϩ , which in the presence of CA drives the reaction towards CO 2 production.…”