Acid-base transport systems ingastrointestinal epithelia

Gut 1992;33: 1134-1145

PROGRESS REPORT

Acid-base transport systems in gastrointestinalepithelia

D Gleeson

Cell plasma membranes contain a variety oftransport systems that carry acid or base into orout of the cell. Research over the past decade hasled to remarkable advances in our understandingof these acid-base transport systems and hasestablished that they have several major physio-logical roles. They are the primary means bywhich cells regulate their internal pH and theyalso contribute importantly to regulation of cellvolume and possibly cell proliferation. Further-more, in the gastrointestinal tract they mediateabsorption and secretion, not only of acids andbases but of several other electrolytes andnutrients.The major acid-base transport systems

on mammalian cell plasma membranes areillustrated in Figures 1-3; their specific locationson different cell types are discussed later.Hydrogen, potassium (H+,K+) adenosine tri-phosphatase (ATPase) extrude H+ in exchangefor K' using energy derived directly from ATPhydrolysis. They are present on the apical mem-branes of gastric parietal cells' (Fig 1) and, insome species, colon epithelial cells,2 where theymediate acid secretion. However, they are notwidely distributed in gastrointestinal epithelia.

Other acid base transport systems derive theirenergy, not directly from ATP hydrolysis, butfrom coupling the movement of one ion topassive movement of another ion along itselectrochemical gradient. In the case of sodium/hydrogen (Na+/H+) exchange (Figs 1-3), H'extrusion from the cell is coupled, in a 1: 1 ratio,to Na+ entry down its chemical gradient.3-' Thisgradient depends on the sodium pump, Na+,K+ATPase, which is present on the basolateralmembrane of all gastrointestinal epithelial cellsand extrudes 3 Na+ ions in exchange for 2 K+ions, thus maintaining a low intracellular [Na+],high intracellular [K'], and negative intra-cellular potential. Na+/H+ exchange is almostubiquitous in mammalian cells and can beinhibited by the diuretic amiloride.There are several mechanisms for bicarbonate

(HCO3 ) transport across plasma membranes.67In most tissues, the enzyme carbonic anhydrasemediates rapid equilibration of H' and HCO3with C02, which diffuses freely across all cellmembranes. Therefore, HCO3 transport intothe cell is equivalent to H` transport out of thecell and vice versa.

In the case of chloride/bicarbonate (Cl-/HC03 ) exchange (Figs 1-3), HCO3 extrusionfrom the cell is coupled, in a 1:1 ratio, to Clentry along its chemical gradient. This gradirnt

H+ HCO3- 3Na+

Figure 1: Acid-base transport systems on an activated gastricparietal cell. The apical membrane contains H+-K+ A TPaseand the basolateral membrane contains Na+lH+ exchange,Cl-IHCO3 exchange, and Na+-HCO3 co-transport. Thestochiometry of the Na+-HCO3 co-transporter is not known(n= 1, 2, or 3). Note also basolateral membrane Na+-K'ATPase and apical membrane Cl- and K+ channels. Fordetails, see text (section 2). As in Figures 2 and 3, the transportsystems which derive their energy directlyfrom ATPhydrolysis, are shown in closed circles.

is a result of the negative intracellular potential,which maintains a low intracellular [Cl -]. In thecase of Na+-HCO3 co-transport (Figs 1, 3),HCO3- movement is coupled with that ofNa+ inthe same direction. In most, although not all,cells this sytem operates in the direction of Na+and HCO3- entry into the cell, the driving force,as for Na+/H+ exchange, being the out to in Na+gradient. Typically, Cl-/HCO3- exchange andNa+-HCO3- co-transport are not affected byamiloride but can be inhibited by the disulphonicstilbene DIDS.6 7 Finally, HCO3- can exit fromsome cells' uncoupled to other ions, via plasmamembrane HCO3- channels (Fig 3). The drivingforce here is the negative intracellular potential.There have been two major approaches to

characterising acid-base transport systems.Firstly, by studying radiolabelled ion uptake

Gastroenterology Unit,Royal HallamshireHospital, SheffieldD GleesonCorrespondence to:Dr D Gleeson,Gastroenterology Unit, FloorJ, Royal Hallamshire Hospital,Glossop Road, SheffieldS10 2JFAccepted for publication4 November 1991

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Acid-base transport systems in gastrointestinal epithelia

H+

Na+

Na+

HCO3--

4~

Ci-

2K+

K+ Cl-H+ 3Na+

Figure 2: Acid-base transport systems on a NaCI absorbingcell. Examples include ileal villus, colon, and gall bladderepithelial cells. The apical membrane contains coupled Na+lH' and Cl-/HCO3 exchangers. The basolateral membranecontains another Na+lH+ exchanger with different kineticcharacteristics and, in addition, Na+-K' ATPase andK'and Cl- channels. In some cells'5 basolateral K' and Cl- exitis via a KCL co-transport system. For details see text (section3).

into isolated plasma membrane vesicles. Forexample, in many tissues, 22Na can be shown tobe concentrated into plasma membrane vesiclesin the presence of an in to out transmembraneH+ gradient. Furthermore, a major componentof this Na+ uptake is a saturable function ofNa+concentration, is temperature sensitive, iselectroneutral (that is, unaffected by trans-membrane potential), and is inhibited byamiloride. These properties are characteristic ofa Na+/H+ exchange mechanism. Isolatedvesicles were used in the first direct demonstra-tion of Na+/H+ exchange, by Murer in rabbitjejunum.9 Similar studies have shown thatnot only Na+/H+ exchange,"-'7 but also,Cl-HCO3- exchange'8 23 and Na+-HCO3-co-transport2425 are widely distributed in gastro-intestinal epithelia from several species includ-ing humans. Furthermore, selective isolation ofvesicles from basolateral and apical membraneshas often revealed a polarised distribution ofthese transport systems between the two mem-branes (Figs 1-3). This polarisation, as discussedbelow, is fundamental to the role of acid-basetransport systems in transepithelial transport.

Acid-base transport systems can also becharacterised in intact cells by their effects onintracellular pH (pHi), an approach facilitated inrecent years by the advent of pH sensitivefluorescent dyes. This approach offers theimportant advantage that the normal environ-ment of the transporter is preserved and hasprovided evidence for regulation of acid-basetransport systems by many hormones, neuro-

transmitters, and growth factors, acting viaintracellular mediators. On the other hand,measurement of pHi using fluorescent dyes hasusually necessitated cell dispersal, followingwhich epithelial cells often lose their polarisedcharacteristics. Consequently, selective assay ofapical and basolateral transport systems hasusually not been possible.

Studies of isolated vesicles and studies of cellpHi, although providing complementary data onacid base transport systems, have defined themin purely functional terms. However, applica-tion of molecular biological techniques hasrecently yielded information regarding themolecular structure of these transport systems.For example, Sardet et al have obtained a humancDNA sequence from human fibroblasts,expression of which restores Na+/H+ exchangeactivity in a mutant fibroblast which lacksintrinsic Na+/H+ exchange.2627 The putativeNa+/H+ exchanger encoded by this cDNAsequence is a 815 amino acid glycoprotein with10-12 hydrophobic (possibly membrane span-ning) domains at its amino terminal. The Cl-/HCO3- exchanger in red blood cells has alsobeen cloned and sequenced.28 Very recently,similar approaches have been used to isolatecDNA sequences encoding Na+/H+ and Cl-/HCO3 exchangers from rabbit ileum (seebelow).

H+ 3Na+Figure 3: Acid-base transport systems on a HCO3- secretingcell. Examples include ileal crypt cells, hepatocytes, and,probably, duodenal epithelial cells and pancreatic and bileductular cells. The apical membrane contains Cl-IHCO3exchange, Cl- channels, and possibly (?), separate HCO3channels andlor Na+-HCO3 co-transport. In order tooperate in the direction ofHCO3 extrusion, the apicalNa+-HCO3 co-transporter would have to transport anegatively charged species, that is n=2 or 3. The basolateralmembrane contdins Na+IH+ exchange andNa+/HCO3 co-transport (the stochiometry ofwhich is not known), togetherwith Na+-K' ATPase. For details see text (section 4).

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Functional roles of acid base transportsystems

(1) REGULATION OF INTRACELLULAR pH (pHi)In most cells, pHi is actively maintained between7.00 and 7.40, almost one unit above the valueexpected if intracellular and extracellular H+were in electrochemical equilibrium. Further-more, pHi can recover spontaneously to baselinewithin minutes after exposure of cells to acuteacid and alkaline loads.67 Regulation of pHi is ofmajor importance to cell homeostasis becausemany physiological phenomena are pH depen-dent29; diverse examples include the rate ofglycolysis,30 the level of intracellular calcium,3'and plasma membrane K' permeability whichmay regulate intracellular electrical potential.2̀3

In most cells, including several gastro-intestinal cells,3"' the major system regulatingpHi, in the absence of HCO3-, is Na+/H+exchange. Under HCO3- free conditions, block-ing the Na+/H+ exchanger with amiloride orremoval of extracellular Na+ typically causes afall in pHi and inhibits recovery from an intra-cellular acid load (usually achieved by pulseexposure of cells to the weak base ammonia).6 7Rapid recovery of pHi after intracellular acidifi-cation is made possible by a distinctive propertyof the Na+/H+ exchanger, first demonstrated inrenal epithelia by Aronson.42 The exchangercontains a modifier site on its intracellular facewhich is activated by H' ions. Thus, intra-cellular acidosis causes a greater increase in Na+/H' exchange activity than that expected fromthe more favourable in to out H' gradient.3 5738Conversely, the Na+/H+ exchanger is downregulated by intracellular alkalosis and is usuallyinactive above a pHi of 7.20, despite iongradients which favour continued Na+/H+exchange. Hormones and growth factors thatregulate the Na+/H+ exchanger often do soby shifting this inverse relationship betweenactivity and pHi 'up' or 'down.' This shift in pHiresponsiveness may be a result of phosphoryla-tion of the exchanger itself, or of intracellularproteins which regulate its activity.327When cells are maintained under more physio-

logical conditions, where HCO3 and CO2 arepresent, other pHi regulatory systems oftenpredominate over Na+/H+ exchange. Forexample, in hepatocytes,' the intestinal cell lineIEC-6,36 partietal cells,43 and pancreatic acinarcells,4' the major mechanism of pHi recoveryfrom an acid load is not affected by amiloride butis inhibited by DIDS; furthermore, it is depen-dent on the presence ofNa+ and HCO3 but notCl -. These properties are characteristic of aNa+-HCO3 co-transport system. In somecells,363744 pHi recovery from an intracellularalkaline load is mediated by HCO3- extrusionwhich is dependent on the presence of Cl- and isinhibited by DIDS, properties characteristic ofthe Cl -/HCO3- exchanger. This exchanger is insome respects a 'mirror image' of Na+/H+exchange in that it possesses an intracellularmodifier site which is sensitive to hydroxyl(OH -) ions2245 1 and thus, although relativelyinactive at baseline pHi, is activated by intra-cellular alkalosis.

In some cells, Cl-/HCO3- exchange is

coupled to the Na+ gradient, which drives it'uphill,' that is Cl- out of and HCO3- into thecell. Na+ dependent Cl-/HCO3- exchange is animportant pHi regulatory mechanism in inverte-brate cells and in mammalian mesenchymalcells6 7 but has not yet been described in gastro-intestinal epithelia.

(2) GASTRIC ACID SECRETION (FIG 1)Gastric acid secretion is mediated by a H+,K+ATPase on the apical membrane of the parietalcell which extrudes H' in exchange for K+.4The enzyme has recently been cloned andsequenced.3' Its molecular weight is about 95 000Daltons and it consists of two subunits: a largeralpha subunit with several membrane spanningdomains and a smaller beta subunit. The struc-tural, enzymatic, and ion transporting propertiesof gastric H+,K+ ATPase have recently beenreviewed. '

In the resting state, H+,K+ ATPase is local-ised, not on the apical membrane of the parietalcell, but in tubulo-vesicular structures in thecytoplasm which are impermeable to K+ andCl-. Thus, its ability to transport H+ into thesevesicles in exchange for K+ is normally limitedby the availability of intravesicular K+. H+transport into vesicles from resting cells can beobserved however, if these vesicles are preloadedwith K+.`' H+ secretion can also be induced byexposure of resting parietal cells to the K+ionophore valinomycin.52

Stimulation of the parietal cell by secreta-gogues such as histamine, gastrin, and carbacholresults in insertion of these H,K+, ATPasecontaining tubulo-vesicles into the apicalmembrane, the area of which increases severalfold.53 55 The permeability of the apical mem-brane to K+ and Cl- also shows a markedincrease. This is partly due to the appearance ofCl- conductive pathways in the apical mem-brane.5158 These pathways are probably Cl-selective ion channels, as demonstrated recentlyby patch-clamp techniques.59 Whether the apicalmembrane K+ permeability increases via open-ing of separate K+ channels,58 6061 as illustrated inFigure 1, or via activation of a KCI co-transportsystem51 7 iS controversial. It is also unclearwhether the increased apical membrane K+ andCl- permeabilities result from activation oflatent transport systems or, alternatively, frominsertion of a separate class of K+ and Cl-permeable cytoplasmic vesicles into the apicalmembrane.62 Whatever the mechanism, theresult is that: (a) the K+ accumulated by H+ ,K+,ATPase can be recycled into the lumen, so thatH+/K+ exchange is no longer limited by avail-ability of K+; and (b) secretion of Cl- ions canaccompany secretion of H+, thereby preservingelectroneutrality.

Omeprazole,63-5 one of the group of substi-tuted benzimidazoles, is a weak base with a pH of4.0. In its uncharged (unprotonated) form, itdiffuses into acid compartments such as theparietal cell lumen, where it becomes protonatedand positively charged. It is thus trapped in theacid compartment, where it accumulates. Theprotonated omeprazole is then converted to anactive metabolite, the sulphenamide, which

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reacts covalently with cysteine residues on theluminal face of the H+,K+ ATPase alpha unit,resulting in irreversible inhibition of H+,K+ATPase and a parallel inhibition of acidsecretion.'By itself, apical HCl secretion would pro-

gressively alkalinise the parietal cell and depleteit of Cl-. However, parietal cell homeostasisseems to be maintained by modulation of threeother acid base transport systems, all localised onthe basolateral membrane: Cl-IHCO3exchange,346768 which lowers pHi by HCO3extrusion and Na+/H+ exchange,3467 68 and Na+-HCO3 co-transport,4369 both of which act toalkalinise the cell. After stimulation of HCIsecretion by histamine, ClW/HCO3- exchangeacross the basolateral membrane increasesseveral fold70'7; the HCO3- efflux represents theblood 'alkaline tide.' This increase in Cl-/HCO3- exchange can be partly ascribed to morefavourable ion gradients, such as the fall inintracellular [Cl-] resulting from apical HCIexit, but it also reflects a true activation of theexchanger. Any rise in pHi resulting from HCIsecretion should activate Cl-/HCO3- exchangevia its OH- sensitive modifier site72; theexchanger may also be directly activated byintracellular mediators, such as cyclic adenosinemonophosphate (AMP) and calcium, whichactivate acid secretion.7' Simultaneously, there isdownregulation of the basolateral transportsystems which act to alkalinise the cell: Na+-HCO3 co-transport73 and, possibly, Na+/H+exchange7' (although this has been disputed70).The net effect of these various modifications is

to oppose the alkalinising effect of apical HCIsecretion, with the result that patietal cell pHiincreases by less than 010 pH units afterstimulation ofHCI secretion.70`7 The parietal cellthus provides a striking example of how, byselective modulation of apical and basolateralacid-base transport systems, it is possible toreconcile the conflicting demands of varyingtransepithelial transport and maintaining cellhomeostasis.The mechanisms by which secretagogues

exert these multiple effects on the parietal cell areincompletely understood. Carbachol, gastrin,and histamine all bind to specific receptors onparietal cell plasma membranes.50 The effects ofcarbachol and gastrin on acid secretion seem tobe mediated via an increase in intracellularcalcium,7475 achieved partly, as in many celltypes, by inducing inositol phosphate turn-over.75"- Histamine also increases intracellularcalcium,7" by a mechanism independent ofinositol phosphate turnover,75` however, itseffect on acid secretion appears to be mediatedmainly via a rise in intracellular cyclic AMP.79"0Calcium, cyclic AMP, and perhaps other media-tors induce phosphorylation of several intracel-lular proteins,"08' which may result in activationof plasma membrane transport systems.

(3) NaCl ABSORPTION (FIG 2)Electroneutral NaCl absorption is a majordriving force for intestinal fluid absorption.02 07Studies in several species, including humans,have shown that electroneutral Na+ and Cl-

absorption in the ileum,"-0 colon,9' 96 and gallbladder059790 are largely interdependent pro-cesses. Furthermore, they are usually inhibitedby amiloride93-%98 " and the carbonic anhydraseinhibitor acetazolamide,0093 94 9 "I' and also(albeit less consistently) inhibited by HCO3--removal959099101 and by DIDS.94 102 These resultslend support to the model originally proposed byTurnberg on the basis of studies in humans,0whereby Na+ and Cl- are transported across theapical membrane via coupled Na+/H+ and Cl-/HCO3 exchange. More recent studies havedirectly confirmed the presence of both Na+/H+and Cl-/IHCO3- exchangers on the apical mem-branes of gall bladder05 and colonic`5 1621epithelial cells and ileal villus cells'4 '9 (Fig 2).The two exchangers seem to be coupledindirectly via changes in pHi'9; a rise in pHiresulting from stimulation of apical Na+/H+exchange activates apical Cl-IHCO3 exchangevia its OH- sensitive modifier site, as recentlyshown in apical membrane vesicles from ileum-6and colon.22 The Na+ accumulated in the cell isthen extruded in exchange for K' by the baso-lateral Na+,K+ ATPase,'03 and the accumulatedCl- leaves the cell via basolateral anion channelsor in some cells05 via a KCI co-transport system.

Intestinal NaCl absorption is regulated byseveral hormones and neurotransmitters.83 84 It isinhibited by agents that increase intracellularcyclic AMP (for example, cholera toxin, prosta-glandin E2, vasoactive intestinal polypeptide(VIP)), intracellular calcium (serotonin, acetyl-choline, substance P), or intracellular cyclicguanosine monophosphate (GMP) (Escherichiacoli toxin).'03-105 This regulation of NaCl absorp-tion may be achieved largely by modulation ofacid-base transport systems. Cyclic AMP, cyclicGMP, and calcium all inhibit Na+/H+ exchangein isolated enterocytes or in ileal apical mem-brane vesicles.10609 Calcium and cylic AMP alsoinhibit both NaCl absorption and apical mem-brane Na+/H1+ exchange in the gall bladder0 ""as do aldosterone"' 112 and activators of proteinkinase C"13'"4 in the colon. In contrast, gluco-corticoids"5 and alpha adrenergic agonists"6"7increase intestinal NaCl absorption; althoughthe effects of these agents on intestinal Na+/H+exchange are not known, both stimulate renalNa+/H+ exchange."8 "9 Intestinal NaClabsorption is also stimulated by respiratoryacidosis99 01 120 and by exposure to volatile fattyacids,'2' interventions which lower pHi andmight thus activate apical membrane Na+/H+exchange via an intracellular H+ sensitivemodifier site. Indeed, volatile fatty acids haverecently been shown to activate apical membraneNa+/H+ exchange in a human colon cancer cellline.'22 Taken together, these studies providestrong evidence that apical membrane Na+/H+exchange is a major regulated step in NaClabsorption. Much less information is availableregarding hormonal regulation of apical Cl-/HCO3- exchange, however the calcium releas-ing agent serotonin inhibits the exchanger in ilealvillus cells'23 as does cyclic AMP in the gallbladder. "0

Intestinal epithelial cells must also maintainhomeostasis in the face of varying rates oftransepithelial NaCl transport. An increase in

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NaCI absorption initiated by stimulation ofapical Na+/H+ exchange and consequent activa-tion of apical Cl-HCO3- exchange via its OH-sensitive modifier site would result not only insome increase in pHi, but also rises in intra-cellular Na+ and Cl- concentrations, cellvolume, and cell membrane potential. Suchdisturbances in cell homeostasis may, as in theparietal cell, be minimised by simultaneousregulation of apical and basolateral membranetransport systems. In gall bladder'24 andcolonic,'25 epithelia, intracellular [Na+] is main-tained virtually constant despite varying rates ofNaCl absorption, suggesting that there is parellelregulation of basolateral Na+/K+ ATPase. Thebasolateral channels which mediate exit of Cl-and exit of the K' accumulated by Na+/K+ATPase are also regulated in parallel with apicalmembrane transport systems in several absorp-tive epithelia. 126

Intestinal acid-base transport systems may besubject to similar coordinated regulation. Recentobservations in isolated vesicles from jejunal," 127ileal,'28-'32 and colonic'5 133 epithelial cells suggestthat the basolateral membrane also possessesa Na+/H+ exchanger. Furthermore, theapical and basolateral exchangers have differentkinetic characteristics; for example, injejunum'27 and ileum'3 129 131 the apical Na+/H+exchanger is relatively resistant to amiloride. Thisdifference between basolateral and apical Na+/H' exchangers was originally shown on a renalepithelial cell line'34 and may be a general propertyof epithelial cells. 134A It is thus possible that thetwo exchangers have distinct functional roles andare selectively regulated. The apical Na+/H+exchanger might be concerned primarily withtransepithelial transport and respond selectivelyto agents which regulate intestinal NaCl absorp-tion. On the other hand, the basolateral Na+/H+1exchanger might be concerned mainly with cellhomeostasis, contributing to regulation of pHiand, possibly also, cell volume and cell prolifera-tion (see below). The apical and basolateralHCO3 transport systems might also havedistinct characteristics and functional roles.Further evaluation of these possibilities awaitsselective study of apical and basolateral acid-basetransport systems and their regulation in intactNaCl absorbing epithelia.

Malabsorption of NaCl as a result of dysfunc-tion of acid-base transport systems may beimportant in the pathogenesis of diarrhoealdisease. The syndrome of diarrhoea with chlori-dorrhoea and alkalosis, seems to result from anabsence of intestinal Cl /HCO3- exchange.'35 136Other cases of congenital diarrhoea have beenascribed to absence of intestinal apical mem-brane Na+/H+ exchange.'37 In several diseases,including cholera, E coli and amoebic infections,bile acid induced diarrhoea, the carcinoid andWerner Morrison syndromes, and inflammatorybowel disease diarrhoea results from disorderedintestinal electrolyte transport caused by avariety of hormones, toxins, and neurotrans-mitters.83 84 103 138~140 These agents may act viaincreases in enterocyte cyclic AMP (for example,cholera toxin, prostaglandin E2, VIP), cyclicGMP (E coli toxin), or calcium (serotonin,substance P, bile acids). The diarrhoea is partly

mediated by intestinal secretion of Cl- andHCO3- (see below), however, malabsorption ofNaCl, via inhibition of apical membrane Na+/H+exchange and possibly Cl-/HCO3- exchange,also plays a role.

(4) HCO3 SECRETION (FIG 3)HCO3- secretion by the stomach, duodenum,and pancreatic and bile ducts is an importantmeans of neutralising gastric acid and of limitingits damaging effects on the mucosa. BiliaryHCO3 secretion may be an important drivingforce for bile acid independent canalicular bileflow,'4' although whether this secretion originatesfrom hepatocytes or from small bile ductules isunclear. HCO3 secretion by the ileum andcolon is an important cause of watery diar-rhoea.86 103 142

In the stomach and duodenum,143ileum, c9144-146 colon,9192'47-149 and pancreas,'50HCO3- secretion is dependent on the presenceof serosal Na+ and is partly dependent on thepresence of Cl- in the lumen. These findings areconsistent with a model involving Na+ depen-dent HCO3 accumulation by the cell across thebasolateral membrane and HCO3 exit acrossthe apical membrane via Cl -/HCO3 exchange,the accumulated Cl- recycling via apical mem-brane Cl- channels.

Such an asymmetrical distribution of trans-port systems has been shown (using isolatedvesicles) in cells from ileal crypts, which seem tobe the site of HCO3- secretion.'5' The apicalmembrane contains Cl-/HCO3 exchange butnot Na+/H+ exchange. This is in contrast to theapical membrane of the absorptive ileal villuscells, which contains both exchangers'52 (com-pare Figs 2 and 3). The ileal crypt cell basolateralmembrane contains two Na+ dependent mecha-nisms for HCO3 accumulation: Na+-HCO3co-transport25 and Na+/H+ exchange,'52 thelatter accumulating HCO3- indirectly via itsalkalinising effect on the cell. Amiloride onlymodestly inhibits ileal HCO3 secretion,'53suggesting that Na+-HCO3 co-transport maybe the more important of the two mechanisms.

Hepatocytes have a similar distribution of acidbase transport systems: basolateral Na+/H+exchange and Na+-HCO3 co-transport,canalicular membrane Cl-/HCO3- exchangeand Cl- channels.'723 '54 ClV/HCO3- exchangeand Cl- channels are also present on the apicalmembranes of duodenal epithelial cells.'55Finally, both Na+/H+ and Cl/HCO33exchange are present in intact pancreatic'56 andbile'57 ductular cells, although in these cells theirlocalisation remains to be established.

In the duodenum'43 and ileum,8645 amajor component of HCO3- secretion isindependent of luminal Cl- and therefore maynot be mediated by apical Cl-/HCO3- exchange.Possible alternative mechanisms for HCO3 exitacross the apical membrane include HCO3permeable channels8 and Na+-HCO3-co-transport. Na+-HCO3- transport has beendiscussed above as a mechanism for cellularaccumulation ofHCO3-, energised by the out toin Na+ gradient. However, in some epithelia,such as the proximal renal tubule,'59 the Na+-

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HCO3- co-transporter has a stochiometry ofthree HCO3- ions for each Na+ ion. It thuscarries a net negative charge and normallyoperates in the direction of HCO3- and Na+extrusion, the driving force being the negativeintracellular potential.HCO3 secretion, like NaCl absorption, is

under elaborate neurohormonal control. It isusually stimulated by agents which increaseintracellular cyclic AMP, for example, secretinin the bile and pancreatic ducts and prosta-glandin E2 in the duodenum.'43 Cholera toxininduces severe diarrhoea by stimulating HCO3-and Cl- secretion throughout the intestine, viaincreases in intracellular cyclic AMP.'03 142 160 161

HCO3- secretion predominates in vivo and Cl-secretion in vitro; the reason for this discrepancyis not known. Leukotriones and prostaglandins,often produced in association with intestinalinflammation, also stimulate HCO3 secre-tion. 162 In contrast, HCO3- secretion in theduodenum and ileum is inhibited by alphaadrenergic agonists. 146 158 163

Mediators such as cyclic AMP might induceHCO3 secretion via several mechanisms. Theseinclude activation of apical membrane Cl-recycling by opening of Cl- channels. CyclicAMP causes opening of apical Cl- channels inseveral secretory epithelia. 1' 165 This effect isabsent in patients with cystic fibrosis,'66-'68 whohave impaired pancreatic HCO3- secretion'69and may possibly have a generalised defect ingastrointestinal HCO3 secretion. Cyclic AMPand/or other mediators might also directlyactivate apical HCO3 exit: (a) via Cl-IHCO3exchange, as demonstrated in duodenal apicalmembrane vesicles"10; (b) via HCO3- channels,as in salivary acinar cells8; or (c) via Na'-HCO3-co-transport.

Alternatively, HCO3 secretion may beinduced via activation of basolateral Na+/H+exchange orNa+-HCO3- co-transport, resultingin cellular HCO3- accumulation and therebyincreasing the driving force for HCO3- exitacross the apical membrane. The accompanyingcell alkalinisation should also activate apical Cl -/HCO3- exchange via its OH- sensitive modifiersite.2246 As discussed in the section on NaClabsorption, cyclic AMP and calcium usuallyinhibit Na+/H' exchange in absorptive epithelia.However, recent studies suggest that serotonin,'23which increases intracellular calcium, andforskolin,'7' which increases intracellular cyclicAMP by direct stimulation of adenylate cyclase,both stimulate the basolateral Na+/H' exchangerin ileal crypt cells. The HCO3 secretagoguecarbachol also activates Na+/H+ exchange insalivary acinar cells.172 Yet another mechanismhas been suggested for secretin induction ofHCO3 uptake into bile ductular cells: insertionof cytoplasmic vesicles containing H+ ATPaseinto the basolateral membrane.'73

Evaluation of these possible mechanismsawaits more detailed characterisation of acid-base transport systems in intact HCO3- secret-ing epithelia. However, by analogy with theparietal cell (see section 2), it seems likely thatHCO3- secretion involves parallel regulation ofboth basolateral and apical HCO3- transportsystems, thereby allowing maintenance of a near

constant pHi through varying rates of trans-epithelial HCO3- transport.

Finally, HCO3- secretion may result not fromdirect activation of acid-base transport systemsbut from movement of weak acids in theirprotonated form in the opposite direction, that isfrom lumen to cell. The net result would beconsumption of luminal H' and therefore (in thepresence of carbonic anhydrase), generation ofHCO3 . Ursodeoxycholic acid'74 and certainother bile acids'75 176 induce a 'hyper' choleresis inseveral species, including man,'77 which resultsmainly from active secretion of HCO3 . ThisHCO3- rich choleresis may partly explain theapparently beneficial effects of ursodeoxycholicacid in cholestatic liver diseases,'76 and also itstendency to induce surface gall stone calcifica-tion, one of the factors limiting its efficacy as acholesterol gall stone dissolving agent.'79

Recent studies involving measurement of bothintracellular and canalicular pH in isolated rathepatocyte couplets suggest that ursodeoxycho-lic acid does not directly activate acid basetransport systems in hepatocytes'80 (althoughan effect on bile ductular cells has yet to beexcluded). An alternative hypothesis has beenproposed, based on the observation that these'hyper' choleretic bile acids are excreted by theliver partly in an unconjugated form.5 176 181According to this 'chole-hepatic shunt' hypothe-sis, the unconjugated bile acid is transported inits ionised form across the hepatocyte canalicularmembrane. Unconjugated bile acids have highpKs, and will thus tend to combine with H'in the bile canaliculus, thereby generating aHCO3 ion. The protonated uncharged (andtherefore lipid soluble) bile acid then recyclesacross -the apical membrane of the bile ductularcell, is returned to the liver, redissociates, andthe bile acid anion is re-excreted. In theory, onebile acid molecule could recycle several times,thereby generating several HCO3- ions in thecanaliculus, and this would explain the 'hyper'choleresis. However, each cycle will result inhepatocyte accumulation of one H' ion which,to maintain the generation of biliary HCO3-,must be extruded across the basolateral mem-brane. The HCO3- rich 'hyper' choleresisinduced by ursodeoxycholic acid is partlyinhibited by amiloride and by Na+ removal,'82suggesting that the hepatocyte basolateralNa+/H+ exchanger'7 contributes to this H'extrusion.

(5) ABSORPTION OF OTHER ELECTROLYTES ANDNUTRIENTS; THE ACID MICROENVIRONMENTThe luminal pH near the intestinal apical mem-brane is maintained at a more acid level than bulkluminal pH. The presence of this acid micro-environment was first suggested by studies in the1950s on intestinal absorption of weak acids,183and has been subsequently confirmed bymicroelectrodes in several species, includingman. 184-188Na+/H+ exchange is present on the apical

membranes of jejunal epithelial cells.9'-" Here, itmay function independently of Cl-/HCO3exchange, the presence of which has not beenconvincingly demonstrated in the jejunum.'` A

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role for apical Na+/H+ exchange in maintainingthe acid microenvironment is suggested byobservations that the microenvironment pH isincreased by luminal perfusion with amiloride orwith Na+ free media,'86 189 although other studieshave failed to confirm these findings.'87 Micro-environment pH is also increased by cyclicAMP, cyclic GMP, and activation of proteinkinase C,'90"'' interventions which, as discussedin section (3), inhibit intestinal Na+/H+exchange. In the ileum and colon, microenviron-ment pH is higher than in the jejunum,'84possibly because the apical Na+/H' exchanger islargely neutralised by parallel Cl-IHCO3-exchange (see section 3 and Fig 2).The acid microenvironment contributes to

intestinal absorption of weak acids. It favoursconversion of these weak acids to their proto-nated uncharged form which can then diffuseacross the apical membrane into the cell.'84 Anexample of this process is HCO3- absorption bythe jejunum and gall bladder. In the humanjejunum, Na+ and HCO3- absorption are inter-dependent, are associated with an increase inintraluminal pCO2, and are inhibited by aceta-zolamide.'92 '3 These observations suggest thatHCO3- is not absorbed directly but rather as aresult of H' secretion via apical Na+/H+exchange. The pCO2 of human gall bladderbile exceeds that of hepatic bile and blood,suggesting that the gall bladder may also absorbHCO3 via H' secretion.'9"'96 Gall bladder H'secretion is impaired in patients with calcified(calcium carbonate rich) gall stones; theresult is an abnormally alkaline gall bladderbile that is supersaturated with calciumcarbonate. 11

Volatile fatty acids (VFAs), are produced bybacterial degradation of non-absorbed dietarycarbohydrate and are rapidly absorbed by thecolon, where they constitute a significant energysource. 84 197 198 In several species, includinghumans,'99-202 colonic VFA absorption in vivo isassociated with HCO3- secretion and a lowerluminal pCO2 than in the absence of VFAs.These findings suggest that VFAs combine withluminal H' (thereby depleting CO2 and generat-ing HCO3 ) and are then absorbed passively intheir protonated (uncharged) form. Otherobservations suggest that the acid microenviron-ment, maintained by apical Na+/H+ exchange,also contributes H' to this process and therebyassists in VFA absorption. For example, VFAabsorption is, like the microenvironment pH,'84relatively independent of bulk luminal pH.'97Furthermore, VFA absorption is associated withenhanced colonic Na+ absorption121 99 200 202 andcan be partly inhibited by amiloride202 and byluminal Na+ removal'99 2"3 A third possiblemechanism for VFA absorption is via a VFA-HCO3 exchange mechanism, recently demon-strated on apical membrane vesicles from humanileum2" and rat colon.205The acid microenvironment also plays a role in

intestinal absorption of folic acid20` and oligopep-tides.207 Patients with coeliac disease or Crohn'sdisease have an abnormally alkaline jejunalmicroenvironment,"'820 which may explain thefrequently observed malabsorption of folic acidand other nutrients in these patients.

(6) REGULATION OF CELL VOLUMEWhen exposed to sudden changes in mediumosmolality, cells behave initially as osmometers,swelling in hypo-osmotic media and shrinking inhyperosmotic media. However, cells then tendto return their volume towards normal, despitecontinued exposure to anisotonic media.209Regulatory volume decrease (RVD) andregulatory volume increase (RVI) are typicallyachieved by activation of plasma membrane iontransport systems, which result ultimately in lossor gain of cell electrolytes and water respectively.There are two major mechanisms for RVI in

mammalian cells. The first mechanism is activa-tion of Na+/H+ exchange with coincidental orsecondary (via a rise in pHi) activation of Cl-/HCO3- exchange, resulting in cell accumulationof NaCl. The accumulated Na+ is extruded byNa+/K+ ATPase in exchange for K' and thusthe net effect is cellular accumulation of KCI.This mechanism seems to be the major meansofRIV in gall bladder210 and some renal2' epithe-lial cells, hepatocytes,22 and lymphocytes.23 Theevidence includes observations that RVI in thesecells is dependent on the presence of Na+, Cl,and HCO3- and furthermore, can be inhibitedby amiloride and by DIDS. In addition, activa-tion of Na+/H+ exchange can be demonstratedby measurement of the effects of hyperosmoticstress on pHi.3209213214 The mechanism(s) ofactivation remain obscure but may involve phos-phorylation of the Na+/H+ exchanger.3The other major mechanism of RVI uses the

out to in Na+ gradient to drive K' and Cl- intothe cell.219 This electroneutral Na+-K-2Cl-co-transport system is present on the basolateralmembranes of intestinal epithelial cells`4 and canbe inhibited by the diuretic bumetanide. It is thissystem, rather than Na+/H+ and Cl-/HCO3-exchange, which seems to mediate RVI in jejunalvillus enterocytes.2' The mechanisms of RVI inother intestinal epithelial cells are not known.

Regulatory volume decrease (RVD) after cellswelling in hypo-osmotic media is mediated byKCI loss, for which there are two main mecha-nisms. The first involves opening of K' and Cl-channels and mediates RVD in hepatocytes2"and jejunal villus enterocytes.26 Other cells maylose KCI via activation of a bumetanide inhibit-ible KCI co-transport system.29The above cell volume regulatory mechanisms

have usually been demonstrated by suddenlyexposing cells to large changes in mediumosmolality. It is not clear to what extent thesemechanisms are operative in vivo, where extra-cellular fluid osmolality is usually regulatedwithin a narrow range. However, for epithelialcells to maintain their volume through varyingrates of transepithelial transport, net ion fluxesacross the apical membrane must balance thoseacross the basolateral membrane. Therefore, cellvolume regulatory mechanisms might also play amajor role in balancing ion fluxes across the twomembranes. Indeed, in some epithelia, theopening of basolateral membrane K' and Cl-channels after an increase in Na+ absorptionacross the apical membrane is mediated partlyvia the resulting increase in cell volume. 16Conversely, carbachol induced stimulation ofHCO3- secretion by salivary acinar cells results

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in activation of basolateral Na+/H+ exchange,which is mediated partly via a decrease in cellvolume,'72 presumably a consequence of apicalHCO3 and water exit.

(7) REGULATION OF CELL PROLIFERATIONOne of the most intriguing and controversialaspects of acid-base transport systems is theirpossible role in the regulation of cellular pro-liferation. In white blood cells, fibroblasts andin various invertebrate cells, several agentswhich induce cell proliferation activate Na/H+exchange5 217; the exchanger is also spontane-ously activated in some tumour cell lines.Furthermore, exposure of cells to amiloride orremoval of Na+ often inhibits their prolifera-tion.5 Based on these observations, it was sug-gested that a rise in pHi resulting from activationof Na+/H+ exchange was a trigger for initiationof cell division.

However, most studies on the effects of mito-gens on pHi have been performed in HCO3 freemedia. Under more physiological conditions,when HCO3 is present, mitogens activate notonly Na+/H+ exchange but also other transportsystems such as Cl-/HCO3 exchange, which insome cells,2829 minimises the changes in pHi.Nevertheless, it is likely that activation of Na+/H' exchange and also the HCO3- transportsystems play a permissive role in cell prolifera-tion by maintaining pHi at a level which willpermit DNA and protein synthesis; these pro-cesses often have pH optima above basal pHi andare inhibited by intracellular acidosis. Indeed, insome cells, simply raising pHi induces prolifera-tion, as was demonstrated recently in fibroblastsby inserting and inducing expression of the genefor H' ATPase.22' It is also possible that acidbase transport systems regulate cell proliferationnot via changes in pHi but via secondary effectson intracellular Na+ or Cl- concentrations or cellvolume.

Gastrointestinal epithelial cell proliferation isinfluenced by many hormonal, paracrine, andintraluminal agents.22' It is still unclear whetheracid-base transport systems are involved inregulating proliferation. However, Na+/H+exchange activity is enhanced in several modelsof increased gastrointestinal cell proliferation,including the neonatal liver,222 the pancreas afterexposure to trophic hormones,223 and theintestine after partial resection224 or exposure tothe carcinogen 1,2 dimethylhydrazine.225 In con-trast, differentiation and cessation of prolifera-tion in some human colon cancer cell lines isassociated with down-regulation of Na+/H+exchange.226 227 Finally, blocking Na+/H+exchange with amiloride or its analogues inhibitsproliferation of a pancreatic cell line and of theliver and jejunum after partial intestinal resec-tion.228229 Further studies in this area will be ofgreat interest.

ConclusionGastrointestinal epithelial cells possess a varietyof acid-base transport systems. The location ofthese transport systems is often highly polarisedbetween the apical and basolateral plasma mem-

branes. They are subject to regulation by manyneural, hormonal, and paracrine factors, actingvia intracellular mediators. There is someevidence that different transport systems, andeven similar transport systems on the apicaland basolateral membranes, are selectivelyregulated. These distinctive properties maypartly explain how acid-base transport systemscan play several major physiological roles,including regulation of pHi and cell volume,transepithelial transport, and, perhaps, cell pro-liferation - roles which might at first seemmutually incompatible.Our understanding of these systems is, how-

ever, still incomplete. For example, the role ofcoordinated regulation of apical and basolateralacid base transport systems in mediatingintestinal NaCl absorption and HCO3 secretionneeds to be systematically evaluated. Thisevaluation will require selective assay of acidbase transport systems on the apical and baso-lateral membranes of polarised gastrointestinalepithelial cells. Such studies have proveddifficult because most epithelia are heterogenousand therefore, measurement of pHi using fluor-escent dyes in one cell type has usually requiredselective cell isolation, following which the cell'spolarised characteristics are often lost.

Recent advances in methodology offer severalpossible solutions to this problem. Firstly,culture of some epithelial cell lines to formmonolayers on semipermeable membranes hasresulted in retention of cell polarity and hasenabled selective study of the apical and baso-lateral membranes of renal'34230 and gastro-intestinal381 22 cell monolayers. Secondly, assay ofacid base transport systems in one cell type in anintact heterogeneous epithelium can sometimesbe achieved by selective loading of pH sensitivedye into that cell type,23' or alternatively by use ofsingle cell fluorescence microscopy.

Yet another advance has been the applicationofmolecular biological techniques to the study ofgastrointestinal acid-base transport systems. Tseet al'3''32 have recently obtained two related butdistinct cDNA sequences from rabbit ileum,which induce expression of Na+/H+ activity ina fibroblast cell line that lacks intrinsic Na+/H+exchange. The first of these putative Na+/H+exchangers has 95% homology with the pre-viously cloned human fibroblast Na+/H+exchanger,262" with which it also shares severalfunctional characteristics, including extremesensitivity to amiloride. Furthermore, immuno-cytochemical studies showed that polyclonalantibodies to a fusion protein incorporating thissequence localise to basolateral but not apicalmembranes from rabbit ileum. The Na+/H+encoded by the second cDNA sequence has beenless extensively characterised but has someproperties common to those of the ileal apicalmembrane Na+/H+ exchanger, including arelative resistance to amiloride. 31 32 Detailedstudies on the neurohormonal regulation of thesetwo putative Na+/H+ exchangers should now bepossible and are awaited with interest. Progresshas also been made in cloning the ileal Cl -/HCO3 exchanger.232Approaches such as these should lead to

further major advances in our understanding of

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these fascinating and versatile transport systemsover the next decade.I am grateful to the Wellcome Trust for financial support and toDr Martin Steward, Department of Physiological Sciences,University of Manchester for critical comment on an earlier draftof the manuscript.

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