H Exchangers and pH Microdomains in Colonic Epithelia

Comp. Biochem. Physiol. Vol. 118A, No. 2, pp. 389–393, 1997 Copyright  1997 Elsevier Science Inc.

ISSN 0300-9629/97/$17.00 PII S0300-9629(96)00326-X

SECTION G: IN VITRO CHARACTERIZATION OF SCFA TRANSPORT

Transepithelial SCFA Gradients Regulate Polarized Na/H Exchangers and pH Microdomains in Colonic Epithelia Marshall H. Montrose and Shaoyou Chu Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, U.S.A.

ABSTRACT. Short chain fatty acids (SCFAs) stimulate electroneutral sodium absorption by activation of apical Na/H exchange in colonocytes. It is often assumed that activation of Na/H exchange is via an intracellular acidification caused by SCFA uptake. These lecture notes review shortcomings in this model of SCFA-stimulated sodium absorption, revealed by recent reports in the literature. This is supplemented by information generated in our laboratory using both a tissue culture model of colonocytes (HT29-C1 cells) and a native tissue preparation (mouse distal colonic mucosa). In both preparations, evidence suggests that physiologic SCFA gradients may generate pH heterogeneity in aqueous microdomains near the plasma membrane of colonocytes. Finally, direct observation of such extracellular microdomains with confocal microscopy is used to support a new model, in which pH microdomains play an important role in regulating both SCFA fluxes and sodium absorption. comp biochem physiol 118A;2:389–393, 1997.  1997 Elsevier Science Inc. KEY WORDS. Carboxy SNARF-1, laser scanning confocal microscope, BCECF, HT29 cells, crypts of Leiberku¨hn, mouse distal colon, pH microclimate

INTRODUCTION Short-chain fatty acids (SCFAs) are present in the colonic lumen due to bacterial fermentation of undigested protein and carbohydrate. A major physiologic function of luminal SCFAs is to activate colonocyte apical Na/H exchange and thereby stimulate electroneutral Na 1 absorption. A similar model of SCFA-stimulated Na 1 absorption has been proposed by many laboratories: SCFA uptake acidifies colonocytes, activates apical Na/H exchange, and thereby stimulates electroneutral Na 1 absorption (1–7). In support of this model, it has been shown that apical Na/H exchange in the colonocyte is activated by intracellular (or intravesicular) acidification (8,9), and SCFAs stimulate Na1 absorption by activation of apical Na/H exchange in colonocytes (2,3, 5,10). Despite significant support for this model of SCFA-stimulated Na 1 absorption, several observations suggest that the model may not be adequate. High concentrations of SCFAs are normally only present at the apical surface of colonocytes, but SCFAs have been added experimentally at different membrane surfaces. If SCFAs can acidify colonocytes via nonionic diffusion H1 monocarboxylate cotransport

Address reprint requests to: Marshall H. Montrose, Ross 930, Johns Hopkins University, 720 Rutland Avenue, Baltimore, MD 21205. Tel. 410-9559681; Fax 410-955-9677. Received 30 May 1996; accepted 31 May 1996.

and/or SCFA/HCO 3 exchange across the basolateral membrane (transport reactions suggested by results from several laboratories), SCFA added at either membrane domain should effectively activate apical Na/H exchange (11,12). However, surprising differences have been found between the stimulation of Na 1 absorption by apical versus basolateral addition of SCFAs. Apical, but not basolateral, SCFAs are able to efficiently stimulate electroneutral Na1 absorption across short-circuited rat distal colon, sheep rumen mucosa, and guinea pig or rabbit gallbladder (2,3,13). To be compatible with the known activation of Na/H exchangers by acidification (9,14), these results suggest that several epithelia either do not acidify in response to basolateral SCFAs, or that SCFA-induced acidification at the basolateral membrane is not sufficient to fully activate apical Na/H exchange. To resolve these discrepancies, we have worked to directly test whether changes in pH explain activation of the polarized Na/H exchangers in response to SCFAs.

RESULTS AND DISCUSSION Role of Intracellular pH in SCFA-Activation of Polarized Na/H Exchangers As summarized below, we have found that homogeneous intracellular acidification cannot explain the activation of polarized Na/H exchangers caused by transepithelial SCFA gradients. Work published in 1994 defined that a physio-

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FIG. 1. Superfusion with basolateral SCFA activates an

amiloride-sensitive pH i recovery mechanism in mouse crypt colonocytes. Muscle-stripped mouse distal colonic mucosa was mounted in a superfusion chamber as described (17), and studied on a digital imaging microscope. Tissue was loaded with SNARF-1 and dual excitation rationing used to quantify intracellular pH (16) of crypt colonocytes within an intact epithelial layer. The presented results are the average response from 20 randomly selected colonocytes in the field of view. The content of superfusates at the luminal (A) and serosal (BL) surfaces of the tissue are indicated. Cells were first equilibrated in a NaCl medium (‘Na’: containing in mM, 130 NaCl, 5 KCl, 1 MgSO4, 2 CaCl2, 20 HEPES, 25 mannose, pH 7.4). At the indicated times the serosal/basolateral superfusate was switched to one containing 130 mM sodium isobutyrate substituted for 130 mM NaCl (SCFA). When indicated, 1 mM amiloride (AMIL) was also present in the basolateral superfusion.

logic (apical-to-basolateral) SCFA gradient was required for selective activation of apical Na/H exchange in a tissue culture model of colonocytes (HT29-C1 cells) (15). More recently, we have used colonic mucosa from mouse distal colon to address the same questions in native tissue. We have loaded crypt colonocytes with SNARF-1 (a pH-sensitive fluorescent dye) and measured intracellular pH (pHi) of single cells in a digital imaging microscope. Previous publications have described the technology for digital ratio imaging of SNARF-1 (16) and mounting of colonic mucosal sheets in a microscope chamber that permits separate superfusion of luminal and serosal surfaces (17). In this native tissue preparation, SCFAs cause cellular acidification of crypt colonocytes. As shown in Fig. 1, addition of 1 mM amiloride (an inhibitor of Na/H exchange) blocks the pHi recovery, which is activated in response to this acidification. This inhibition can also be observed as a dimunition of the alkaline pHi overshoot caused when SCFA is subsequently removed. We have shown previously that this overshoot is a consequence of net proton efflux during pHi recovery (16), in this case, due to Na/H exchange. Results in Fig. 1 suggest that basolateral SCFA activates basolateral Na/H exchange. As shown in Fig. 2, both apical and basolateral SCFA acidify crypt colonocytes. At equimolar [SCFA], cellular acidification due to apical SCFA is less than basolateral

M. H. Montrose and S. Chu

SCFA. This can be due to intrinsic differences in SCFA permeability at the different membranes (31), and/or limited nonionic SCFA uptake due to luminal extracellular alkalinization (17). However, even when SCFA concentrations are biased to produce equivalent cellular acidification in response to either apical or basolateral SCFA, there is a marked polarity in the cellular response. The hallmarks of active Na/H exchange (pHi recovery alkalinization during SCFA exposure, and alkaline overshoot when SCFA is removed) are less in the presence of apical vs basolateral SCFA. Results suggest that only basolateral SCFA is effectively stimulating basolateral Na/H exchange. Although both apical and basolateral Na/H exchange are likely to contribute to the pHi recovery alkalinization (see Chu and Montrose in this issue), the basolateral Na/H exchange predominates after SCFA exposure. Results in Fig. 2 show that selective activation of polarized Na/H exchangers occurs in native tissue. Thus similar to HT29-C1 cells (15), homogeneous intracellular acidification cannot explain selective activation of polarized Na/H exchangers by physiologicallyoriented SCFA gradients. This implies that physiologic SCFA gradients activate Na/H exchange by previously unanticipated mechanisms. As summarized below, the mechanism in question is unlikely to involve allosteric interactions between SCFAs and Na/H exchanger proteins, or to require SCFA metabolism. When HT29-C1 cells were acidified to a similar extent by either bilateral propionate or ammonium-prepulse, Na/H exchange rates were indistinguishable (15). This suggested that it was the change in pH that was important for transport activation, and not direct SCFA-protein interactions or SCFA metabolism. In addition, reversing orientation of the SCFA gradient affected transport activation (although pHi was again invariant), an effect difficult to ascribe to

FIG. 2. Polarized pH i regulation in response to apical vs ba-

solateral addition of SCFA. Experiments were performed as described in Fig. 1. Perfusates had the same composition as described in Fig. 1 legend, except that one perfusate (16 SCFA) had only 16 mM sodium iso-butyrate (substituted for 16 mM NaCl in Na medium).

Transepithelial SCFA Gradients

SCFA metabolism. Thus, activation of polarized Na/H exchangers cannot be explained by homogeneous acidification of the cytosol or generation of a SCFA metabolite, since neither event should distinguish between different orientations of the transepithelial SCFA gradient. We have tested the hypothesis that transepithelial SCFA gradients may generate pH heterogeneity in aqueous microdomains near the plasma membrane. pH Heterogeneity in the Colonic Epithelium Based on the information reviewed above, it became increasingly important to measure pH with high spatial resolution in the colonic epithelium. If the response to SCFAs was more complex than a homogeneous intracellular acidification, the additional information could help resolve controversies about mechanisms of SCFA transport, and SCFA regulation of colonic Na/H exchange. It has been shown that pHi can be heterogeneous within single cells, and that optical methods can detect such heterogeneity. In plant cells, pHi gradients were shown to be important in tip growth (18). In fibroblasts, localized application of the SCFA propionate was shown to generate localized acidification of the cytoplasm (19). Work from several laboratories suggest that SCFAs may also change pH in extracellular domains impinging on the colonic epithelium. Previous work suggested that pH at the apical and/or basolateral extracellular surface of colonocytes may not be at equilibrium with pH in the bulk phase. At the apical extracellular surface of surface colonocytes, a pH microclimate has been proposed to modify SCFA transport by affecting protonation of SCFAs (20). In a related observation, propionate affected surface pH near the hen colonic mucosa (but not in rat or guinea pig) (21,22). Unfortunately, the physiologic relevance of this concept has remained controversial, because of technical limitations in these pH electrode measurements (poorly defined spatial resolution, poorly buffered, and unstirred medium). Recent information suggests that optical probes have adequate resolution to detect acidic pH microdomains in the basolateral extracellular surface of cultured renal epithelia (23,24). The presence of such a basolateral pH microdomain has been predicted by mathematical modelling of transepithelial SCFA fluxes in the intestine, which concluded that pH disequilibrium should exist in an extracellular basolateral compartment (25,26). Thus, the use of optical probes has been adequate to detect both intracellular and extracellular pH microdomains, which are believed to mediate physiologic regulation of membrane transport. Visualization of Extracellular pH Microdomains Our recent work with confocal microscopy has succeeded in visualizing extracellular pH near mouse crypt colonocytes with high spatial resolution, and has evaluated the tissue response to SCFAs (17). We used a Bio-Rad MRC-600 con-

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focal microscope to measure the fluorescence of carboxy SNARF-1 free acid (a pH-sensitive dye), when the dye was added to superfusates bathing mouse colonic epithelium. This form of SNARF-1 is cell-impermeant and so its fluorescence can, in theory, report pH in all spaces surrounding crypt colonocytes. However, we first had to address previous findings (27) that fluorescence signal was attenuated by light scatter from the tissue, an effect that diminished confocal signal as a function of focal distance into the tissue. Fortunately, we were able to use a dye that is pH-insensitive (Lucifer Yellow) to verify that light scatter losses were precisely equivalent at the required emission wavelengths (Fig. 3). This implied that dual-emission fluorescence ratios were able to correct for light scatter, so that calibration curves of ratio vs pH could be used legitimately for confocal pH determination. Using confocal measurements of pH in native tissue, we have shown that SCFAs cause localized extracellular pH changes. Physiologically-oriented SCFA gradients simultaneously alkalinized the crypt lumen and acidified lamina propria (17). In particular, the pH in the crypt lumen becomes 0.5–0.8 pH units more alkaline than the bulk superfusate, despite physical continuity between the bulk and crypt luminal solutions. We have shown that restricted access of solutions into the crypt lumen permit steady-state maintenance of this pH disequilibrium (17). However, the underlying vectorial proton fluxes that are the basis for polarized pH regulation have not yet been fully defined, and could be due to nonionic diffusion and/or carrier-mediated SCFA transport. For illustration, a simple non-ionic diffusion model is shown in Fig. 4. As shown in the model, fueling a sustained apical-to-basolateral SCFA flux may cause net consumption of apical protons and production of basolateral protons in the extracellular environments. Extracellular pH regulation is observed throughout the lower half of the mouse colonic crypt (the full range of our measurement capabilities) (28). Experiments used the confocal to collect images along 40-µm of the crypt-to-surface axis (starting from the base of the crypt and working toward the surface). Apical SCFAs caused a luminal alkalinization that was maximal 10–20 µm from the crypt base and then of diminished magnitude nearer the crypt neck. The maximal acidification of the lamina propria occurred nearer the surface than the luminal alkalinization (28). Since diffusion should equilibrate such gradients over a few sec, results suggested that significant variations in net cellular proton flux along the crypt-to-surface axis must be invoked to explain the observed luminal pH variation. The polarized changes in extracellular pH can qualitatively explain the observed polarized activation of Na/H exchangers. Since Na 1 and H1 compete for the extracellular transport site of Na/H exchangers, extracellular alkalinization is predicted to activate Na/H exchange at the apical membrane by increasing affinity for extracellular sodium (14). This would lead to significant acceleration of transport in an environment where [Na] is below Kt, such as in the

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FIG. 4. Model for how transepithelial nonionic diffusion of SCFA could generate heterogeneity of pH across an epithelial cell. The model depicts an apical-to-basolateral SCFA gradient, and the arrows indicate the direction of net flux of both membrane transport and chemical equilibrium. The arrows are not meant to imply that reactions are irreversible.

FIG. 3. Light scattering diminishes confocal fluorescence sig-

nal as a function of focal depth into colonic tissue. Tissue was imaged with 1003 water objective, and a BioRad MRC600 confocal microscope used to collect Z-series of images (17) while tissue was superfused at all surfaces with NaCl medium containing either 0.1 mM SNARF-1 (A), or 0.2 mM Lucifer Yellow (B). A region of subepithelial tissue was randomly selected for analysis. Fluorescence at 640 nm (m,d) or 580 nm (n,s) was quantified in this region from each image in a Z-series. Values were normalized to the fluorescence observed at the most shallow optical section, and plotted vs Z-axis focal distance into tissue. Data points in the figure are mean 6 SEM of 4 experiments. For each data set, results were fit to a single exponential. The best fit lines are presented for both 640 nm (⋅⋅⋅⋅), and 580 nm results (——). The exponential decay rate constants are similar for SNARF-1 fluorescence (640 nm 5 0.030 mm21; 580 nm 5 0.028 mm21) and Lucifer Yellow fluorescence (640 5 0.029 mm21; 580 5 0.027 mm21).

colonic lumen. Conversely, acidification of lamina propria could inhibit basolateral Na/H exchange by raising Kt(Na). Thus, transepithelial SCFA gradients and observed extracellular pH changes are sufficient to explain polarized activation of Na/H exchange. However, it must be stated that there may be other sites of pH heterogeneity. As shown in Fig. 4, local heterogeneity of pHi is also predicted and pHi gradients have been reported in other cells (18,19). This has not yet been evaluated for colonocytes. However, the heterogeneity of pHi predicted in Fig. 4 would also cause polarized activation of Na/H exchangers in the appropriate orientation to explain observations. Alternatively, it is also possible that changes in cell volume (33) may contribute to the selective activation of exchangers, but this has not yet been tested. Independent of any contribution from pHi gradients or other untested factors, evidence already strongly suggests that pH heterogeneity will overall be an important component in the selective activation of colonocyte Na/H exchangers. In summary, SCFAs regulate both intracellular and extracellular pH in colonic epithelia, and extracellular pH regulation is implicated in the selective activation of polarized Na/H exchangers. We speculate that extracellular pH regulation may have other important roles in the physiologic actions of SCFAs. For instance, SCFAs have been shown to stimulate ammonium absorption and bicarbonate secretion (29,30). A transepithelial pH gradient should act as a driving force for the vectorial titration of these weak acid/bases across the epithelium. The luminal alkalinization may also act as a feedback control on net uptake of SCFAs. Luminal alkalinization will reduce the concentration of the non-ionized SCFA form and proportionally reduce SCFA uptake via non-ionic diffusion. These and other functions of extracellular pH regulation need to be explored in further work. However, it already seems clear that the ability to visualize events along the crypt-to-surface axis of living tissue (28) will greatly expand our understanding of colonic epithelial function.

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