The enzyme carbonic anhydrase (CA), responsible for catalysing CO2 hydration (Roughton, 1964), is differentially expressed in many mammalian cells (Dodgson, 1991) raising questions regarding its physiological functions. Carbonic anhydrase is known to play a central role in intracellular buffering and CO2 transport in red blood cells (Meldrum & Roughton, 1932), in acid secretion by the stomach (Davenport, 1939) and in the regulation of intracellular (Roos & Boron, 1981) and extracellular pH (Chen & Chesler, 1992). In contrast, in the small intestine, where expression and activity of CA isozymes vary along the length of the intestinal tract, its key function remains uncertain (Carter & Parsons, 1972; Fleming et al. 1995). In the present work we have used carboxy-SNARF-1 to image intracellular pH (pHi) confocally in isolated small intestinal enterocytes, and we have studied the effects on local cytoplasmic pH of activating an H+-coupled peptide transporter located at the apical pole of the cell (Ogihara et al. 1996). This transporter provides the principal route for intestinal absorption of the peptide products of protein digestion. Our results suggest a novel role for CA in controlling pHi non-uniformities that are induced in the enterocyte by this localized acid influx.
- 1The spatial and temporal distribution of intracellular H+ ions in response to activation of a proton-coupled dipeptide transporter localized at the apical pole of mouse small intestinal isolated enterocytes was investigated using intracellular carboxy-SNARF-1 fluorescence in combination with whole-cell microspectrofluorimetry or confocal microscopy.
- 2In Hepes-buffered Tyrode solution, application of the dipeptide Phe-Ala (10 mM) to a single enterocyte reduced pHi locally in the apical submembranous space. After a short delay (8 s), a fall of pHi occurred more slowly at the basal pole.
- 3In the presence of CO2/HCO3−-buffered Tyrode solution, the apical and basal rates of acidification were not significantly different and the time delay was reduced to 1 s or less.
- 4Following application of the carbonic anhydrase inhibitor acetazolamide (100 μM) in the presence of CO2/HCO3− buffer, addition of Phe-Ala once again produced a localized apical acidification that took 5 s to reach the basal pole. Basal acidification was slower than at the apical pole.
- 5We conclude that acid influx due to proton-coupled dipeptide transport can lead to intracellular pH gradients and that intracellular carbonic anhydrase activity, by facilitating cytoplasmic H+ mobility, limits their magnitude and duration.
Isolation of mouse enterocytes
Epithelial cells were isolated from the small intestine of 4- to 8-week-old mice killed by cervical dislocation, by a combination of enzymatic (0.5 mg ml−1 hyaluronidase; Sigma) and mechanical dispersion (Kimmich, 1970). The cells were finally suspended in CO2/HCO3−-buffered medium and kept on ice (∼4°C) until use.
Hepes-buffered Tyrode solution contained (mM): NaCl, 140; KCl, 4.5; MgCl2, 1; CaCl2, 2; D-mannose, 10; and Hepes, 20; adjusted to pH 7.4 with 4 M NaOH at 37°C. Bicarbonate-buffered Tyrode solution was identical except that the NaCl concentration was reduced to 117 mM and 23 mM NaHCO3 was substituted for Hepes. Bicarbonate solutions were equilibrated with 5 % CO2/95 % air for ∼2 h prior to experiments (pH 7.4, 37°C). Dipeptides were added as solids and solution pH was adjusted to 7.4 at 37°C with 4 M NaOH. Acetazolamide was obtained from Sigma.
Intracellular pH was measured ratiometrically in mouse isolated enterocytes using the pH-sensitive fluorophore carboxy-SNARF-1 (Molecular Probes, Eugene, OR, USA). Isolated enterocytes were incubated in a 5-10 μM solution of the acetoxymethyl (AM) ester in Hepes-buffered Tyrode solution (14 min, 25°C). Carboxy-SNARF-1-loaded cells were excited at 540 ± 12 nm and emission was measured simultaneously at 590 ± 5 and 640 ± 5 nm with a customized (Buckler & Vaughan-Jones, 1990) inverted epifluorescence microscope (Nikon Diaphot). Signals were then digitized at 0.5 kHz (CED 1401). The emission ratio 590 nm: 640 nm was calculated and converted to pH values using the established nigericin in situ calibration method (Thomas et al. 1979). Rates of change of pHi (dpHi/dt) at any given pHi were obtained by computer from the first-time differential of the best-fit polynomial equation (SigmaPlot, SPCC) to experimental data points sampled at 0.5 s intervals.
Carboxy-SNARF-1-loaded cell imaging was performed at 37°C on a Leica DM IRBE inverted microscope with Leica TCS NT software, using a Leica × 100 NA 1.4 oil-immersion, planoapochromat objective lens. SNARF excitation was achieved with the 514 nm laser line of an air-cooled 25 mW argon laser. A ratiometric image was generated every 2.8 s from the fluorescence emission images collected using two photomultiplier tubes with 640 nm and 580 ± 20 nm band-pass filters (Glen Spectra, Middlesex, UK). Image analysis was performed off-line using the NIH Image analysis software program (available at http://rsb.info.nih.gov/nih-image/). Ratio pixel values (R)were converted to pH values using the equation:
where pKa, Rmin and Rmax were determined using an in situ pH calibration curve generated under similar conditions to the microspectrofluorimetric calibration experiments. Fmin and Fmax are the minimum and maximum fluorescence values determined at 640 nm emission for low and high pH, respectively (Buckler & Vaughan-Jones, 1990).
Local pHi was measured from ratiometric values in pre-defined regions of interest (20 pixels × 20 pixels). Data values (means ± standard deviation) were transferred to Sigma Plot for further analysis.
In order to measure the time delay for a fall in pHi at the apical compared with the basal pole, a threshold value was chosen (i.e. 0.04 pH units). This was approximately equal to twice the standard deviation (2 × 0.017 ± 0.001 pH units, n= 14) of the noise in the recording of resting pHi. Therefore, the moment of first appearance of intracellular acidosis in a defined region was taken as the time at which a fall of 0.04 pH units could be detected (see e.g. Fig. 3A).
Intracellular carboxy-SNARF-1 concentration
In rabbit ventricular myocytes the intracellular mobility of carboxy-SNARF-1 is ∼2.8 × 10−7 cm s−1 (R. D. Vaughan-Jones & K. W. Spitzer, unpublished data). In the present study the intracellular concentration of carboxy-SNARF-1 ([carboxy-SNARF-1]i) was estimated by superfusing AM-loaded enterocytes with varying concentrations of cell impermeant dextran-conjugated carboxy-SNARF-1 and measuring, at a common pHo/pHi of 7.40, fluorescence intensity at 640 nm within equal areas of interest inside/outside the cell. When the two intensities are equal then [carboxy-SNARF-1]i=[carboxy-SNARF-1]o, assuming no significant difference in the spectral properties of intracellular and extracellular SNARF. Using this method, [carboxy-SNARF-1]i was found to be ∼100 μM. In view of the large intrinsic buffering capacity in isolated enterocytes (i.e. 20-30 mmol l−1; authors’ unpublished data), and assuming the low SNARF-1i mobility found in myocytes, the low [SNARF-1]i will not significantly affect intracellular H+ mobility.
All statistical data are expressed as means ±s.e.m. and n is the sample size.
Dipeptide-induced acid loading
Figure 1A shows the effects of proton-coupled dipeptide transport on pHi in mouse isolated enterocytes superfused with Hepes-buffered Tyrode solution (pHo 7.4). The pHi was recorded using whole-cell microspectrofluorimetry and is thus an average value for intracellular pH in all regions of the cell. In isolated enterocytes pHi was 7.38 ± 0.01 (n= 60), consistent with previous reports (Isenberg et al. 1993). Upon adding the dipeptide phenylalanyl alanine (Phe-Ala) (10 mM), an intracellular acidification occurred (0.16 ± 0.09 pH units at 0.239 ± 0.02 pH units min−1, n= 38). The initial rate of intracellular acidification (see inset to Fig. 1A), when plotted as a function of dipeptide concentration, displayed saturation kinetics (Fig. 1A), as expected for a proton-coupled peptide transporter (Thwaites et al. 1993). Comparable rates of acidification were observed upon addition of 10 mM glycyl-alanine (Fig. 1C, n= 27) whereas there was no significant acidification upon application of the constituent amino acids L-alanine (10 mM) or glycine (10 mM) (n= 5,P > 0.05, Student's unpaired t test) (Fig. 1A). Thus the acidification was a direct result of H+-coupled transport of the intact peptide and not of the products of peptide hydrolysis.
Imaging intracellular pH
A confocal microscope set in reflectance scan mode was used to view the morphology of individual enterocytes. The nucleus was identified as the dark area located towards the basal pole while the white area at the other pole identified the brush-border membrane (Fig. 2A). Confocal imaging of carboxy-SNARF-1-loaded isolated enterocytes revealed a cytoplasmic fluorescence which, when measured at each emission wavelength (i.e. 590 or 640 nm), revealed no significant difference in intensity between apical and basolateral poles. Longer dye incubation protocols (> 20 min) led to sequestration of the dye within intracellular organelles; consequently, loading times were kept to a minimum. Resting pHi in isolated enterocytes, determined from the average whole-cell pHi signal, was 7.36 ± 0.03 pH units (n= 31) in Hepes-buffered Tyrode solution (pHo 7.4, 37°C), in good agreement with pHi measured under similar experimental conditions using conventional microspectrofluorimetry (7.38 ± 0.01 pH units, n= 60).
The spatial and temporal distribution of pH was imaged following a brief application (∼1 min) of Phe-Ala (10 mM) to the superfusate (Fig. 2A). An inwardly directed flow of acidification was observed at the intracellular face of the apical membrane (indicated by a red arrow) which then spread towards the basal pole. This is shown more clearly in Fig. 2A where mean resting fluorescence intensity from control images displayed in Fig. 2A has been subtracted from subsequent images, to emphasize the pHi fall induced by Phe-Ala. The maximal rate of acidification measured (see Methods) at the apical end was 0.380 ± 0.057 pH units min−1 compared with 0.275 ± 0.042 pH units min−1 at the basolateral end (rates compared at a common mean pHi of 7.27 ± 0.10, n= 7,P < 0.05, Student's paired t test). This suggests that H+ influx across the apical membrane was occurring at a rate faster than the subsequent intracellular diffusion of protons along the apical-basal axis of the cell. A time delay between the onset of acidification at the apical and basal poles was readily observed in all experiments in Hepes-buffered Tyrode solution and amounted to 7.6 ± 1.0 s (n= 7). It is unlikely that transmembrane acid efflux would have affected this significantly since the time delay is 16-fold smaller than the average half-time for transporter-mediated whole-cell pHi recovery from intracellular acidosis (t0.5= 126.4 ± 9.6 s, n= 13). While we do not exclude entirely some influence of acid extruders on the time delay, it is more likely to be a function of intracellular rather than transmembrane H+ movement.
The temporal aspects of proton gradient formation are explored more extensively in Fig. 3, one of seven numerical analyses. The orientation of the enterocyte and the position of the apical pole are displayed in Fig. 3A. Plots of intracellular acidification after adding 10 mM Phe-Ala are displayed in Fig. 3A for three locations within the cell: apical, middle and basolateral. In addition, Fig. 3A shows 3-dimensional plots of the spatial distribution of pHi as a function of time. It is clear that the rise of [H+]i occurred initially at the apical pole with a proton gradient developing along the length of the cell so that the concentration change at the basal end occurred with a delay and at a significantly slower rate (n= 7,P < 0.05, Student's paired t test).
The functional presence of carbonic anhydrase
We wished to test if formation of a pHi gradient could occur in CO2/HCO3−-buffered conditions. We therefore initially investigated if CA activity was expressed in the cells since this controls the efficiency of intracellular CO2-dependent buffering (Roos & Boron, 1981; Leem & Vaughan-Jones, 1998). Two conventional, microfluorimetric recordings of pHi are superimposed in Fig. 4A. Upon changing the superfusate from Hepes to a 5 % CO2/HCO3−-buffered Tyrode solution (pH 7.4), there was a rapid, reversible acidification of ∼0.21 pH units, which was slowed (by 50 ± 2 %, n= 6,P < 0.05, Student's paired t test) in the presence of the membrane-permeant CA inhibitor acetazolamide (ACTZ). Steady-state pHi in CO2/HCO3− was unaffected by ACTZ (n= 6,P < 0.05, Student's paired t test). In further photometry experiments using CO2/HCO3−-buffered medium, 100 μM ACTZ increased the initial rate of acidification produced with Phe-Ala (10 mM) by 70 ± 23 % (P < 0.05,n= 4), whereas there was no effect of ACTZ in Hepes-buffered medium (91.4 ± 10.8 %; P > 0.05,n= 5). These data confirm the presence of functional CA activity in this cell (Carter, 1972).
Spatial and temporal intracellular pH gradients in CO2/HCO3−
In confocal images, the fall of pHi in the apical region of interest (0.14 ± 0.03 pH units) and the initial rate of apical acidification (0.188 ± 0.017 pH units min−1) were significantly less than in Hepes (reduced by ∼35 % and ∼50 %, respectively, n= 4,P < 0.05, Student's unpaired t test) consistent with the increase of intracellular buffering power in CO2/HCO3− (Roos & Boron, 1981; Leem & Vaughan-Jones, 1998). More importantly, there was no detectable time delay for the appearance of acidification at the basal pole. Moreover, formation of an apical-basal pHi gradient was not evident (Fig. 4A). It should be noted, however, that the time resolution of the measurements was set by the pHi image-collection frequency (0.36 Hz) so that a brief, transient occurrence of pHi non-uniformity between successive frames (2.8 s) could not be excluded. Indeed when the threshold for detecting basal acidosis was increased from ΔpHi= 0.04 (see Methods) to ΔpHi= 0.06, then in three of the four experiments, a small time delay of 1.2 ± 0.5 s was apparent (detected by interpolating between successive measurement points). It is clear, nevertheless, that the pHi non-uniformity that is readily induced in Hepes buffer is greatly reduced in CO2/HCO3− buffer.
Importance of carbonic anhydrase to pHi gradient formation
Addition of acetazolamide (100 μM) in the presence of HCO3−-buffered medium reversed much of the effect of CO2/HCO3− on the apical-basal pHi gradient. Thus in CO2/HCO3− medium, application of 10 mM Phe-Ala in the presence of acetazolamide reduced pHi where the initial fall was faster apically than basally (0.126 ± 0.018 vs. 0.073 ± 0.017 pH units min−1, respectively; rates compared at common mean pHi, n= 5,P < 0.05, Student's paired t test). This resulted in the formation of an apical-basal pHi gradient as illustrated in Fig. 4A (see panels taken at 15 and 55 s). Furthermore, a significant time delay between acidification at apical and basal poles was now detectable (5.2 ± 1.4 s, n= 5,P < 0.05, Student's paired t test).
It should be noted in Fig. 4A that the initial pHi gradient induced by Phe-Ala was replaced after 90 s by a more uniform elevation of [H+]i. A similar result was observed in three out of the five experiments in CO2/HCO3− plus ACTZ. The previous experiments in Hepes-buffered media were not sufficiently long to discover if pHi uniformity was eventually established but the non-uniformity in Hepes could certainly extend for at least 2.5 min (see e.g. Fig. 3A).
Apparent intracellular H+ diffusion coefficient
We have adapted the Einstein equation (eqn (2)) to approximate the apparent intracellular H+ diffusion coefficient (Dapp):
where t is the time delay between apical and basal acidification, and x is the distance between the mid-points of the apical and basal regions of interest.
Mean values for Dapp have been plotted in the histogram shown in Fig. 4D. Note that in order to estimate Dapp in CO2/HCO3− in the absence of ACTZ, we used the small time delay detected at threshold ΔpHi= 0.06 rather than 0.04. This will give a lower limit for Dapp under these conditions. It appears that the presence of a CO2/HCO3− buffer system increases Dapp by at least 4-fold, while adding ACTZ subsequently reduces it to a value comparable to that seen in the absence of CO2/HCO3−.
In the present study we have shown a novel role for carbonic anhydrase (CA), namely that of facilitating intracellular acid diffusion, thus helping to dissipate localized intracellular pH gradients in mouse small intestinal enterocytes. In the absence of CA activity (e.g. in Hepes media, or in CO2/HCO3−-buffered media containing ACTZ) a physiological acid influx across the apical membrane produces significant non-uniformity of pHi. To our knowledge these are the first experimental demonstrations of a transport-induced pHi gradient in a eukaryotic cell although such intracellular pH gradients have been hypothesized and modelled in various cell types (Junge & McLaughlin, 1987; Irving et al. 1990; al Baldawi & Abercrombie, 1992). In the presence of CO2/HCO3−, but with no CA inhibitor present, the pHi non-uniformity is greatly attenuated, thus illustrating the important influence of CA activity.
In an unbuffered solution, the movement of acid depends on the diffusion coefficient for H+ and/or OH−. In the present work we cannot distinguish between H+ or counter OH− movement or between the effects of hydronium or hydroxonium ions. For convenience, we have defined these movements in terms of intracellular H+ mobility. As pointed out by Junge & McLaughlin (1987), the cytoplasmic movement of H+ is likely to depend less on its free mobility than on the mobility and dissociation of the intracellular buffers since these are present at much higher concentrations and reversibly bind almost all intracellular acid. Because their physical size is considerably larger than that of the H+ ion, they will be less mobile so that the overall H+i mobility should be lower than in a simple unbuffered solution. This is confirmed by our measurements of H+ mobility (Dapp) within the enterocyte. When employing Hepes-buffered superfusates, our value for Dapp is 1000-fold lower than for H+ in pure water (i.e 8-9 × 10−5 cm2 s−1; Longsworth, 1954). Interestingly, our value is also 20-fold smaller than a previous estimate of Dapp in extruded Myxicola axoplasm (Irving et al. 1990; al Baldawi & Abercrombie, 1992) and smaller than that calculated theoretically for frog skeletal muscle cytoplasm (Irving et al. 1990). The H+ mobility reported for extruded axoplasm may be higher than our present value because of the unusually high endogenous concentrations (> 100 mM) in Myxicola of amino acids such as glycine that may act as relatively mobile buffers.
Our finding that acid movement is enhanced in the presence of CO2/HCO3− is consistent with a higher mobility for the intracellular components of the CO2-dependent buffer (i.e. HCO3−, CO2 and H2CO3) relative to the overall mobility of the intrinsic buffers. Adding even small quantities of mobile buffer to a solution of poorly mobile buffer (such as intrinsic proteins) can greatly increase overall H+ mobility (Junge & McLaughlin, 1987). The present results, however, do not identify the components of the CO2 buffer that limit this increase. One possibility is that the limit is set by the internal HCO3− mobility, although our estimate of H+i mobility under these conditions (Fig. 4A) is over 30-fold lower than that quoted for HCO3− in free solution (1.185 × 10−5 cm2 s−1; Lide, 1995). An important assumption here is that the reversible hydration of CO2 to produce HCO3− and H+ is essentially instantaneous within cytoplasm and therefore not rate limiting. While this buffer reaction is catalysed by intracellular CA, the functional expression of CA in the enterocyte is only modest (Fig. 4A). We therefore do not exclude the possibility that our present estimate for intracellular H+ mobility in CO2/HCO3−-buffered conditions may be limited by the speed of the buffer reaction itself. It is notable that ACTZ slows the chemical buffer step by 50 % (Fig. 4A) and that this reduces H+ mobility by 3-fold. It is unlikely that ACTZ interferes directly with the cytoplasmic diffusion of bicarbonate (or indeed of CO2 or H2CO3). We must therefore conclude that intracellular CA activity, by regulating the kinetics of reversible CO2 hydration, facilitates the speed of H+ movement within the enterocyte.
The conclusion that CA isoform activity in enterocytes helps to dissipate intracellular proton gradients by facilitating diffusive H+ movement has important nutritional implications. Specifically, the enhanced movement of H+ away from the intracellular sub-apical space will help to optimize the chemical driving force for proton-coupled peptide transport, thus increasing the efficiency of absorption of the oligopeptide products of lumenal protein digestion. The present study also raises more general questions regarding a similar functional role for CA in other mammalian cells, including neurones and glia in the central nervous system where localized acid movements can occur (Bouvier et al. 1992; Chesler & Kaila, 1992).
This work was funded by a studentship (to A. K. Stewart) from the Medical Research Council and an equipment grant (to R. D. Vaughan-Jones) from The Wellcome Trust.